Unnamed: 0
int64 0
1.42k
| context
stringlengths 2.88k
70.8k
| document_id
int64 185
2.68k
| question
stringlengths 11
194
| answers
dict | id
int64 225
5.32k
|
|---|---|---|---|---|---|
800 | Host resilience to emerging coronaviruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/
SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4
Authors: Jamieson, Amanda M
Date: 2016-07-01
DOI: 10.2217/fvl-2016-0060
License: cc-by
Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome.
Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] .
In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care.
Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses.
Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] .
The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] .
The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] .
Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] .
One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] .
Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge.
Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] .
REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.
A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV.
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome.
• Antivirals have limited effects on the course of the infection with these coronaviruses.
• There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus.
• Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health.
• Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience.
• The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients.
Papers of special note have been highlighted as: | 1,671 | Which medical comorbidities most profoundly influenced MERS-CoV outcomes? | {
"answer_start": [
13213
],
"text": [
"if they were obese, immunocompromised, diabetic or had cardiac disease"
]
} | 1,279 |
801 | Host resilience to emerging coronaviruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/
SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4
Authors: Jamieson, Amanda M
Date: 2016-07-01
DOI: 10.2217/fvl-2016-0060
License: cc-by
Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome.
Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] .
In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care.
Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses.
Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] .
The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] .
The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] .
Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] .
One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] .
Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge.
Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] .
REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.
A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV.
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome.
• Antivirals have limited effects on the course of the infection with these coronaviruses.
• There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus.
• Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health.
• Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience.
• The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients.
Papers of special note have been highlighted as: | 1,671 | Which immune factors were associated with increased SARS-CoV morbidity and mortality? | {
"answer_start": [
13463
],
"text": [
"a higher neutrophil count and low T-cell counts"
]
} | 1,280 |
802 | Host resilience to emerging coronaviruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/
SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4
Authors: Jamieson, Amanda M
Date: 2016-07-01
DOI: 10.2217/fvl-2016-0060
License: cc-by
Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome.
Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] .
In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care.
Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses.
Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] .
The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] .
The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] .
Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] .
One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] .
Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge.
Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] .
REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.
A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV.
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome.
• Antivirals have limited effects on the course of the infection with these coronaviruses.
• There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus.
• Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health.
• Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience.
• The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients.
Papers of special note have been highlighted as: | 1,671 | What is the prognostic role of coinfection in SARS-CoV and MERS-CoV infections? | {
"answer_start": [
13525
],
"text": [
"One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria"
]
} | 1,281 |
803 | Host resilience to emerging coronaviruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/
SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4
Authors: Jamieson, Amanda M
Date: 2016-07-01
DOI: 10.2217/fvl-2016-0060
License: cc-by
Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome.
Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] .
In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care.
Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses.
Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] .
The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] .
The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] .
Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] .
One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] .
Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge.
Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] .
REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.
A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV.
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome.
• Antivirals have limited effects on the course of the infection with these coronaviruses.
• There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus.
• Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health.
• Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience.
• The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients.
Papers of special note have been highlighted as: | 1,671 | Can host resilience be predicted? | {
"answer_start": [
13735
],
"text": [
"A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted"
]
} | 1,282 |
804 | Host resilience to emerging coronaviruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/
SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4
Authors: Jamieson, Amanda M
Date: 2016-07-01
DOI: 10.2217/fvl-2016-0060
License: cc-by
Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome.
Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] .
In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care.
Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses.
Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] .
The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] .
The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] .
Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] .
One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] .
Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge.
Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] .
REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately.
A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV.
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome.
• Antivirals have limited effects on the course of the infection with these coronaviruses.
• There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus.
• Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health.
• Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience.
• The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients.
Papers of special note have been highlighted as: | 1,671 | Can biomarkers be used to predict outcomes in acute respiratory distress (ARDS) patients? | {
"answer_start": [
13874
],
"text": [
"Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients"
]
} | 1,283 |
805 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | What is required for a Hepatitis B infection in cells? | {
"answer_start": [
1158
],
"text": [
"both intracellular and cell-surface factors"
]
} | 2,996 |
806 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection? | {
"answer_start": [
1727
],
"text": [
"heparan sulfates in the membrane proteins"
]
} | 2,997 |
807 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | Which protein domain of the Hepatitis B envelope is necessary for infection? | {
"answer_start": [
2168
],
"text": [
"Nterminus of HBV preS1 (amino acids 1-47) "
]
} | 2,998 |
808 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | Where is NTCP located in the body? | {
"answer_start": [
4322
],
"text": [
"lateral surface (canalicular) of hepatocytes"
]
} | 2,999 |
809 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | What does the NTCP protein mediate? | {
"answer_start": [
4383
],
"text": [
"bile acid transport "
]
} | 3,000 |
810 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | Is NTCP sufficient to allow HBV infection? | {
"answer_start": [
5012
],
"text": [
"not sufficient"
]
} | 3,001 |
811 | One step closer to an experimental infection system for Hepatitis B Virus? --- the identification of sodium taurocholate cotransporting peptide as a viral receptor
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562259/
SHA: f4f36a8e9fee64d59ccf22b724c7dab345102658
Authors: Chen, Pei-Jer; Wu, T-C
Date: 2013-01-11
DOI: 10.1186/2045-3701-3-2
License: cc-by
Abstract: Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research.
Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane.
The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).
HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.
Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV.
Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide.
NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system.
Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.
The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment. | 1,552 | Why is NTCP thought to not be sufficient for HBV infection? | {
"answer_start": [
5054
],
"text": [
"the majority of HepaRG cells were found to express NPCT but not to be infected"
]
} | 3,002 |
812 | Knowledge, Attitudes and Practices (KAP) related to the Pandemic (H1N1) 2009 among Chinese General Population: a Telephone Survey
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112099/
SHA: fe954b75ed45c02d47090ee70d25c726b24b081c
Authors: Lin, Yilan; Huang, Lijuan; Nie, Shaofa; Liu, Zengyan; Yu, Hongjie; Yan, Weirong; Xu, Yihua
Date: 2011-05-16
DOI: 10.1186/1471-2334-11-128
License: cc-by
Abstract: BACKGROUND: China is at greatest risk of the Pandemic (H1N1) 2009 due to its huge population and high residential density. The unclear comprehension and negative attitudes towards the emerging infectious disease among general population may lead to unnecessary worry and even panic. The objective of this study was to investigate the Chinese public response to H1N1 pandemic and provide baseline data to develop public education campaigns in response to future outbreaks. METHODS: A close-ended questionnaire developed by the Chinese Center for Disease Control and Prevention was applied to assess the knowledge, attitudes and practices (KAP) of pandemic (H1N1) 2009 among 10,669 responders recruited from seven urban and two rural areas of China sampled by using the probability proportional to size (PPS) method. RESULTS: 30.0% respondents were not clear whether food spread H1N1 virusand. 65.7% reported that the pandemic had no impact on their life. The immunization rates of the seasonal flu and H1N1vaccine were 7.5% and 10.8%, respectively. Farmers and those with lower education level were less likely to know the main transmission route (cough or talk face to face). Female and those with college and above education had higher perception of risk and more compliance with preventive behaviors. Relationships between knowledge and risk perception (OR = 1.69; 95%CI 1.54-1.86), and knowledge and practices (OR = 1.57; 95%CI 1.42-1.73) were found among the study subjects. With regard to the behavior of taking up A/H1N1 vaccination, there are several related factors found in the current study population, including the perception of life disturbed (OR = 1.29; 95%CI 1.11-1.50), the safety of A/H1N1 vaccine (OR = 0.07; 95%CI 0.04-0.11), the knowledge of free vaccination policy (OR = 7.20; 95%CI 5.91-8.78), the state's priority vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), and taking up seasonal influenza vaccine behavior (OR = 4.69; 95%CI 3.53-6.23). CONCLUSIONS: This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account.
Text: At the end of March 2009, an outbreak of novel influenza A (H1N1) (here after called A/H1N1) infection occurred in Mexico, followed by ongoing spread to all over the world in a short period [1] . On June 11 2009, the World Health Organization raised its pandemic alert level to the highest level, phase 6 [2] , meaning that the A/H1N1 flu had spread in more than two continents and reached pandemic proportions. As of June 13, 2010, it had caused over 18,172 deaths in more than 214 countries and overseas territories or communities [3] . Most illness, especially the severe illness and deaths, had occurred among healthy young adults, which was markedly different from the disease pattern seen during epidemics of seasonal influenza [4, 5] .
China is highly susceptible to A/H1N1 because of its huge population and high residential density, besides the high infectiousness of this novel influenza virus. After the first imported case reported on May 11, 2009 , the confirmed cases were reported in various provinces of China [6] . By the late of October 2009, A/H1N1 cases had increased dramatically, with 44,981 cases and 6 deaths confirmed at the end of October 2009. The A/ H1N1 infection rate peaked in November 2009, when approximately 1500 new cases of A/H1N1 were being confirmed each day. By the end of this month, a total of 92,904 cases and 200 deaths had resulted from A/ H1N1-related causes [7] . The Chinese government has taken a series of preventive measures according to WHO guidelines, including the promotion of public knowledge about flu through mass media, patient isolation, quarantine of close contact person, and free vaccinations to population at high risk (e.g. young children, healthcare workers, and people with chronic disease) [8] . However, there were few public reports on the assessment of the effect of these policies and the level of knowledge, attitude and practice (KAP) associating with A/H1N1 among general population.
It is well-known that confused comprehension and negative attitude towards the emerging communicable disease may lead to unnecessary worry and chaos, even excessive panic which would aggravate the disease epidemic [9] . For instance, during SARS epidemic from 2002 to 2004, the misconceptions and the excessive panic of Chinese public to SARS led the public resistant to comply with the suggested preventive measures such as avoiding public transportation, going to hospital when they were sick, which contributed to the rapid spread of SARS and resulted in a more serious epidemic situation, making China one of the worst affected countries with over 5327 cases and 439 deaths [10, 11] . In addition, the panic of infectious disease outbreak could cause huge economic loss, for example the economic loss of SARS has been estimated at $30-$100 billion in US, though less than 10,000 persons were infected [12] . SARS experience has demonstrated the importance of monitoring the public perception in disease epidemic control, which may affect the compliance of community to the precautionary strategies. Understanding related factors affecting people to undertake precautionary behavior may also help decision-makers take appropriate measures to promote individual or community health. Therefore, it is important to monitor and analyze the public response to the emerging disease.
To investigate community responses to A/H1N1 in China, we conducted this telephone survey to describe the knowledge, attitudes and practices of A/H1N1 among general population in China and put forward policy recommendations to government in case of future similar conditions.
This study was performed in seven urban regions (Beijing, Shanghai, Wuhan, Jingzhou, Xi'an, Zhengzhou, Shenzhen cities) and two rural areas (Jingzhou and Zhengzhou counties) of China with over one million people in each region. Regarding the urban sites, Beijing as the capital of China locates in the northeast; Shanghai is a municipality in the east of China; Wuhan (the provincial capital of Hubei) and Zhengzhou (the provincial capital of Henan province) are both in the centre of China; Xi'an in the northwest of China is the provincial capital of Shanxi province; and Shenzhen of the Guangdong province is in the southeast of China. As for the rural sites, Jingzhou county and Zhengzhou county, from Hubei and Henan provinces, respectively, both locate in the centre of China.
This current study was carried out in three phases during the pandemic peak season of A/H1N1. The first phase was from 30 November 2009 to 27 December 2009, the second from 4 January 2010 to 24 January 2010, and the third from 24 February to 25 March in 2010.
A two-stage proportional probability to size (PPS) sampling method was used in each phase. In stage І, about 30% of administrative regions in each study site were selected as primary sample units (PSUs) for cluster sampling. In stage II, telephone numbers were sampled randomly, of which the first four digitals were obtained from each PSU's post office as initial number and the other three or four digitals were obtained from random number generated by Excel 2003. Then each family was chosen as per unit (excluding school, hotel public or cell phone etc.) and at least 400 families in each site at each phase were selected finally. If the family was selected repeatedly or refused to answer the questionnaire, we added one to the last digit of phone number and dial again. If the line was busy or of no response, we would dial three times and then give up this phone number if there was still no respondent.
Anonymous telephone interviews were conducted from 6:30 pm to 10:00 pm so as to avoid over-presenting the non-work population by well-trained interviewers with Bachelor degree of Epidemiology. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of A/H1N1 Pandemic by Telephone was designed by the Chinese Centre for Disease Control and Prevention (China CDC, Beijing). The majority of the questions were closed-ended and variables in the questionnaire were categorical, except age. The inclusion criteria of subjects were: age≥18 and proper communication skills. There were seven questions related to the knowledge of A/H1N1, four referred to the attitude, and five concerning about the practice in this questionnaire (See additional file 1: The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China).
This study was approved by the institutional review board of the Tongji Medical College of Huazhong University of Science and Technology. All respondents were informed consent. We respected their wishes whether to accept our survey and promised to protect their secrets.
All data were entered into computer using Epidata V.3.1 and were analyzed in SPSS statistical software V.12. Chi-square test was applied to compare the immunization rates of the seasonal flu and A/H1N1 vaccine. The associations between the socio-demographic factors and the KAP regarding A/H1N1 were firstly investigated by using univariate odds ratios (OR) and then stepwise logistic regression modeling applied. Adjusting for such background variables including gender, age, level of education, occupation, region, and survey wave, stepwise multivariate logistic regression models were applied to investigate the impact factors associated with the risk perception of A/H1N1, A/H1N1 vaccination uptake and the compliance with suggested preventive measures (avoid crowd places/wash hand frequently/keep distance from people with influenza-like symptoms). For the purposes of analysis, the factor knowledge about the main modes of transmission was divided into two groups according to whether the respondents knew both cough and talk faceto-face can spread A/H1N1. Odds ratios and respective 95% confidence intervals (CI) were obtained from the logistic regression analysis. P values lower than 0.05 were judged to be statistically significant.
A total of 88541 telephone numbers were dialed. Except 65323 invalid calls (including vacant numbers, fax numbers, busy tone numbers and non-qualified respondents whose age <18 and whose phones were from school, hotel or other public places), 23218 eligible respondents were identified. Among these respondents, 12360 completed the interview. Therefore, the response rate was 46.8%. Excluding missing, and logical erroneous data, 10669 questionnaires in total were eligible for analysis. The baseline characteristics of the respondents were presented in Table1. The mean age of all respondents was 41.47 years (over range: 18-90 year) . Of all respondents, 54.4% were female, and 42.4% had received college or above education (Table 1) .
The overall KAP related to A/H1N1 was reported in Table 2 . As to knowledge, 75.6% of all respondents knew that influenza could be transmitted by coughing and sneezing, and 61.9% thought that talking face-to-face was the transmission route, whereas 30.0% believed the transmission could be through food. Less than one third of respondents knew that virus could be transmitted by handshaking and indirect hand contact (26.8% and 22.3%, respectively). Multiple logistic regression analysis showed that those with middle school (OR = 1.71; 95%CI 1.48-1.98), or having an education level of college and above (OR = 2.16; 95%CI 1.83-2.54) were more likely to know the transmission routes comparing with other people. Comparing with students, teachers (OR = 1.46; 95%CI 1.09-1.96) were more likely to answer the above questions Table 3 and Table 4 ). Regarding the A/H1N1vaccination, 69.9% respondents believed that the occurrence rate of adverse reactions caused by A/H1N1 vaccination was fairly low and they were not afraid of taking up vaccination. Most residents (96.1%) thought that the state's vaccination strategy was reasonable.
About half of the respondents (42.9%) had avoided going to crowded places during the past two weeks of our survey. In case people nearby held influenza-like symptoms such as fever or cough, 56.9% increased the frequency of hand-washing and 57.4% would stay away from them. Multiple logistic regression analysis indicated compliance with the preventive practices were more likely to be taken by those who were females (OR = 1. Table 3 and Table 4 ). The immunization rates of the seasonal flu and A/ H1N1 in respondents were 7.5% and 10.8% respectively. The multivariate stepwise models further showed that except the health care workers (OR = 1.52; 95%CI 1.09-2.11), residents in other occupations (OR = 0.06-0.67) were less likely to take up the A/H1N1 vaccination comparing with students (in Table 3 ). Adjusting for the background covariates the knowledge about the free vaccination policy (OR = 7.20; 95%CI 5.91-8.78) and the state's initial vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), perception of daily life disturbed (OR = 1.29; 95% CI 1.11-1.50), practice of injecting the seasonal influenza vaccine (OR = 4.69; 95%CI 3.53-6.23) were significantly associated with behavior of taking up the A/H1N1 vaccination positively (in Table 5 ), and the adverse reaction of A/H1N1 vaccine negatively influenced people's practice (OR = 0.07; 95%CI 0.04-0.11).
Novel A/H1N1 has caused pandemic in this century. It is important to encourage the public to adopt precautionary behaviors, which is based on the correct knowledge of the epidemic and appropriate response among residents. Many studies have examined the various levels of KAP about infectious disease outbreaks, such as SARS, avian influenza [13] [14] [15] . Some studies have been reported specifically on community responses to A/H1N1 in Australia and Europe [16, 17] . But through literature search, we haven't found any public reports on KAP regarding A/H1N1 among Chinese population until now. Therefore, we conducted this large population-based survey (10669 respondents) to investigate community responses to A/H1N1 and to provide baseline data to government for preventive measures in case of future outbreaks.
Unless people have basic knowledge about the modes of transmission, they respond appropriately during an outbreak [16] . It has been proved that influenza is transmitted through person to person via respiratory secretions [18] . Most residents in our survey recognized that OR m : odds ratio obtained from stepwise multivariate logistics regression analysis using univariately significant variables as candidate variables and adjusting for region; NU: not significant in the univariate analysis; *: P < 0.05; †: P < 0.01; ‡: P < 0.0001.
the risk of getting infected would increase when an infected person coughed or sneezed in close distance. This may be due to the previous experience of SARS and avian flu. Multivariate analysis results showed that workers and farmers with lower education level were less likely to have this knowledge, which indicated that the contents and forms of propaganda should be more understandable and acceptable. A large proportion of residents in our survey overlooked the indirect hand contact and hand-shaking transmission route and about one third of public misconceived that A/H1N1 was food borne, which was associated with the previous knowledge of avian flu and the new A/H1N1 flu in the general population. The confusion with avian flu might mislead some residents to believe that the A/H1N1 virus is fatal and cause public panic [19] . Therefore, it is important for the government and health authorities to provide continuously updated information of the emerging disease through televisions, newspapers, radios, and Internet. There are regional differences in the perception of A/H1N1. For example, the public in Hong Kong did not perceive a high likelihood of having a local A/H1N1 outbreak [19] , but Malaysians were particularly anxious about the pandemic [20] . The current study shows that emotional distress was relatively mild in China as few residents worried about being infected (25.1%). This phenomenon may also be related to the previous experience of the SARS epidemic, as well as the open epidemic information. A survey in Korean university showed that women perceived higher illness severity and personal susceptibility to A/ H1N1 infection, which had been reconfirmed in our study [21] . Logistic regression analysis results suggested that women with higher educational level had higher perception of risk. As time went by, the knowledge about the main transmission route increased, but the risk perception of being infected in residents decreased, suggesting the positive effect of government policy regarding A/H1N1 infection prevention, as well as the promotion of the media.
The previous study presented various results of influencing factors on the the compliance with the preventive practices. The study in Saudi showed that older men with better education were more likely to take preventive practices [9] ; female students in Korean washed hands more frequently during the peak pandemic period of A/ H1N1 [21] ; in another pandemic study in USA, younger people was found to have greater uptake of recommended behaviors but not for gender [16] . We found female with higher education took more precautionary behaviors, but office staffs and farmers took less comparing with students. While such differences could result from study population demographics, profound differences may also exist in the knowledge of A/H1N1 and the perceptions of recommended behaviors in those countries. Adjusting for the background factors, the multivariate logistic regression showed the possible relationship between knowledge and risk perception, knowledge and practices (odd ratios were 1.57 and 2.09, respectively), which indicated that good knowledge is important to enable individuals to have better attitudes and practices in influenza risk reduction. Similar findings were observed in other studies performed during A/ H1N1 pandemic in Singapore [22] and during SARS pandemic in Hong Kong [13] . Therefore, it is important to focus on inculcating the correct knowledge to individuals as it will influence both attitudes and practices. Injecting vaccination is an effective measure to prevent infectious disease [23] . In China, the seasonal influenza vaccination is not included in the national immunization program and must be purchased by recipients. Those who are above 60 years old, the pupil and children in kindergarten, and people with chronic diseases are recommended to get inoculation. Data provided by China CDC in 2009 showed that the immunization rate of the seasonal flu in Chinese population was below 2% [24] , which was much lower than 7.5% in our study (P < 0.0001). This phenomenon is partly due to the state's prior vaccination strategy for population at high risk such as students, teachers, healthcare workers and people with chronic disease, as well as the confusion between seasonal flu vaccine and A/H1N1 vaccine in residents. People who couldn't access the A/H1N1 vaccine may take up seasonal flu vaccine as preventive behaviors. The A/ H1N1 vaccine was not available in China until the middle of September 2009. All populations at high risk above three years old were invited for vaccination free of charge [25] . A survey among 868 European travelers showed 14.2% participants were vaccinated against pandemic influenza A/H1N1 [26] , higher than 10.8% in our study (P < 0.01). Our study also showed students and health care workers were more likely to take up, which may be due to the prior vaccination strategy. Multivariate stepwise logistic regression analysis, which allowed us to adjust for background factors, further showed the perceived risk of infection and the knowledge about the main modes of transmission related to A/H1N1 vaccination were insignificantly, similar results seen in Lau's study [8] . Therefore, the vaccination rate of A/H1N1 is not expected to increase even if the virus becomes more prevalent or the knowledge of its transmission mode improved. Additionally, the behavior of taking up A/H1N1 vaccine was associated with perceptions of vaccine's safety and influence on daily life by A/H1N1 as well as the knowledge about the free vaccination policy and the state's initial vaccination strategy. This suggests that improving the safety of vaccine, the acceptability of side effect and the knowledge about the state's strategy related to A/H1N1 vaccination in residents may be helpful to promote A/H1N1 vaccination in the general population. The cross-sectional telephone survey adopted in the study has some limitations. We were unable to interview the people who did not have phones and the depth of the questionnaire was largely limited because questions and pre-existing answers could not be too long and complex. In addition, the telephone response rate was 46.8%, which means more than half of the interviewees rejected or didn't finish the survey. It was impossible to compare the difference between respondents and nonrespondents due to the lack of their basic information.
This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic as most of them confused the transmission route of A/H1N1. There are many factors influencing the KAP related to A/H1N1. Female with higher educational level had higher perceived risk of infection and took more precautionary behaviors. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account. The data collected in this survey could be used as baseline data to monitor public perceives and behaviors in the event of future outbreak of infectious disease in China.
Additional file 1: Questionnaire. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China. | 2,675 | In 2009 what was the reported H1N1 vaccination rate in China? | {
"answer_start": [
1433
],
"text": [
"10.8%"
]
} | 523 |
813 | Knowledge, Attitudes and Practices (KAP) related to the Pandemic (H1N1) 2009 among Chinese General Population: a Telephone Survey
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112099/
SHA: fe954b75ed45c02d47090ee70d25c726b24b081c
Authors: Lin, Yilan; Huang, Lijuan; Nie, Shaofa; Liu, Zengyan; Yu, Hongjie; Yan, Weirong; Xu, Yihua
Date: 2011-05-16
DOI: 10.1186/1471-2334-11-128
License: cc-by
Abstract: BACKGROUND: China is at greatest risk of the Pandemic (H1N1) 2009 due to its huge population and high residential density. The unclear comprehension and negative attitudes towards the emerging infectious disease among general population may lead to unnecessary worry and even panic. The objective of this study was to investigate the Chinese public response to H1N1 pandemic and provide baseline data to develop public education campaigns in response to future outbreaks. METHODS: A close-ended questionnaire developed by the Chinese Center for Disease Control and Prevention was applied to assess the knowledge, attitudes and practices (KAP) of pandemic (H1N1) 2009 among 10,669 responders recruited from seven urban and two rural areas of China sampled by using the probability proportional to size (PPS) method. RESULTS: 30.0% respondents were not clear whether food spread H1N1 virusand. 65.7% reported that the pandemic had no impact on their life. The immunization rates of the seasonal flu and H1N1vaccine were 7.5% and 10.8%, respectively. Farmers and those with lower education level were less likely to know the main transmission route (cough or talk face to face). Female and those with college and above education had higher perception of risk and more compliance with preventive behaviors. Relationships between knowledge and risk perception (OR = 1.69; 95%CI 1.54-1.86), and knowledge and practices (OR = 1.57; 95%CI 1.42-1.73) were found among the study subjects. With regard to the behavior of taking up A/H1N1 vaccination, there are several related factors found in the current study population, including the perception of life disturbed (OR = 1.29; 95%CI 1.11-1.50), the safety of A/H1N1 vaccine (OR = 0.07; 95%CI 0.04-0.11), the knowledge of free vaccination policy (OR = 7.20; 95%CI 5.91-8.78), the state's priority vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), and taking up seasonal influenza vaccine behavior (OR = 4.69; 95%CI 3.53-6.23). CONCLUSIONS: This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account.
Text: At the end of March 2009, an outbreak of novel influenza A (H1N1) (here after called A/H1N1) infection occurred in Mexico, followed by ongoing spread to all over the world in a short period [1] . On June 11 2009, the World Health Organization raised its pandemic alert level to the highest level, phase 6 [2] , meaning that the A/H1N1 flu had spread in more than two continents and reached pandemic proportions. As of June 13, 2010, it had caused over 18,172 deaths in more than 214 countries and overseas territories or communities [3] . Most illness, especially the severe illness and deaths, had occurred among healthy young adults, which was markedly different from the disease pattern seen during epidemics of seasonal influenza [4, 5] .
China is highly susceptible to A/H1N1 because of its huge population and high residential density, besides the high infectiousness of this novel influenza virus. After the first imported case reported on May 11, 2009 , the confirmed cases were reported in various provinces of China [6] . By the late of October 2009, A/H1N1 cases had increased dramatically, with 44,981 cases and 6 deaths confirmed at the end of October 2009. The A/ H1N1 infection rate peaked in November 2009, when approximately 1500 new cases of A/H1N1 were being confirmed each day. By the end of this month, a total of 92,904 cases and 200 deaths had resulted from A/ H1N1-related causes [7] . The Chinese government has taken a series of preventive measures according to WHO guidelines, including the promotion of public knowledge about flu through mass media, patient isolation, quarantine of close contact person, and free vaccinations to population at high risk (e.g. young children, healthcare workers, and people with chronic disease) [8] . However, there were few public reports on the assessment of the effect of these policies and the level of knowledge, attitude and practice (KAP) associating with A/H1N1 among general population.
It is well-known that confused comprehension and negative attitude towards the emerging communicable disease may lead to unnecessary worry and chaos, even excessive panic which would aggravate the disease epidemic [9] . For instance, during SARS epidemic from 2002 to 2004, the misconceptions and the excessive panic of Chinese public to SARS led the public resistant to comply with the suggested preventive measures such as avoiding public transportation, going to hospital when they were sick, which contributed to the rapid spread of SARS and resulted in a more serious epidemic situation, making China one of the worst affected countries with over 5327 cases and 439 deaths [10, 11] . In addition, the panic of infectious disease outbreak could cause huge economic loss, for example the economic loss of SARS has been estimated at $30-$100 billion in US, though less than 10,000 persons were infected [12] . SARS experience has demonstrated the importance of monitoring the public perception in disease epidemic control, which may affect the compliance of community to the precautionary strategies. Understanding related factors affecting people to undertake precautionary behavior may also help decision-makers take appropriate measures to promote individual or community health. Therefore, it is important to monitor and analyze the public response to the emerging disease.
To investigate community responses to A/H1N1 in China, we conducted this telephone survey to describe the knowledge, attitudes and practices of A/H1N1 among general population in China and put forward policy recommendations to government in case of future similar conditions.
This study was performed in seven urban regions (Beijing, Shanghai, Wuhan, Jingzhou, Xi'an, Zhengzhou, Shenzhen cities) and two rural areas (Jingzhou and Zhengzhou counties) of China with over one million people in each region. Regarding the urban sites, Beijing as the capital of China locates in the northeast; Shanghai is a municipality in the east of China; Wuhan (the provincial capital of Hubei) and Zhengzhou (the provincial capital of Henan province) are both in the centre of China; Xi'an in the northwest of China is the provincial capital of Shanxi province; and Shenzhen of the Guangdong province is in the southeast of China. As for the rural sites, Jingzhou county and Zhengzhou county, from Hubei and Henan provinces, respectively, both locate in the centre of China.
This current study was carried out in three phases during the pandemic peak season of A/H1N1. The first phase was from 30 November 2009 to 27 December 2009, the second from 4 January 2010 to 24 January 2010, and the third from 24 February to 25 March in 2010.
A two-stage proportional probability to size (PPS) sampling method was used in each phase. In stage І, about 30% of administrative regions in each study site were selected as primary sample units (PSUs) for cluster sampling. In stage II, telephone numbers were sampled randomly, of which the first four digitals were obtained from each PSU's post office as initial number and the other three or four digitals were obtained from random number generated by Excel 2003. Then each family was chosen as per unit (excluding school, hotel public or cell phone etc.) and at least 400 families in each site at each phase were selected finally. If the family was selected repeatedly or refused to answer the questionnaire, we added one to the last digit of phone number and dial again. If the line was busy or of no response, we would dial three times and then give up this phone number if there was still no respondent.
Anonymous telephone interviews were conducted from 6:30 pm to 10:00 pm so as to avoid over-presenting the non-work population by well-trained interviewers with Bachelor degree of Epidemiology. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of A/H1N1 Pandemic by Telephone was designed by the Chinese Centre for Disease Control and Prevention (China CDC, Beijing). The majority of the questions were closed-ended and variables in the questionnaire were categorical, except age. The inclusion criteria of subjects were: age≥18 and proper communication skills. There were seven questions related to the knowledge of A/H1N1, four referred to the attitude, and five concerning about the practice in this questionnaire (See additional file 1: The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China).
This study was approved by the institutional review board of the Tongji Medical College of Huazhong University of Science and Technology. All respondents were informed consent. We respected their wishes whether to accept our survey and promised to protect their secrets.
All data were entered into computer using Epidata V.3.1 and were analyzed in SPSS statistical software V.12. Chi-square test was applied to compare the immunization rates of the seasonal flu and A/H1N1 vaccine. The associations between the socio-demographic factors and the KAP regarding A/H1N1 were firstly investigated by using univariate odds ratios (OR) and then stepwise logistic regression modeling applied. Adjusting for such background variables including gender, age, level of education, occupation, region, and survey wave, stepwise multivariate logistic regression models were applied to investigate the impact factors associated with the risk perception of A/H1N1, A/H1N1 vaccination uptake and the compliance with suggested preventive measures (avoid crowd places/wash hand frequently/keep distance from people with influenza-like symptoms). For the purposes of analysis, the factor knowledge about the main modes of transmission was divided into two groups according to whether the respondents knew both cough and talk faceto-face can spread A/H1N1. Odds ratios and respective 95% confidence intervals (CI) were obtained from the logistic regression analysis. P values lower than 0.05 were judged to be statistically significant.
A total of 88541 telephone numbers were dialed. Except 65323 invalid calls (including vacant numbers, fax numbers, busy tone numbers and non-qualified respondents whose age <18 and whose phones were from school, hotel or other public places), 23218 eligible respondents were identified. Among these respondents, 12360 completed the interview. Therefore, the response rate was 46.8%. Excluding missing, and logical erroneous data, 10669 questionnaires in total were eligible for analysis. The baseline characteristics of the respondents were presented in Table1. The mean age of all respondents was 41.47 years (over range: 18-90 year) . Of all respondents, 54.4% were female, and 42.4% had received college or above education (Table 1) .
The overall KAP related to A/H1N1 was reported in Table 2 . As to knowledge, 75.6% of all respondents knew that influenza could be transmitted by coughing and sneezing, and 61.9% thought that talking face-to-face was the transmission route, whereas 30.0% believed the transmission could be through food. Less than one third of respondents knew that virus could be transmitted by handshaking and indirect hand contact (26.8% and 22.3%, respectively). Multiple logistic regression analysis showed that those with middle school (OR = 1.71; 95%CI 1.48-1.98), or having an education level of college and above (OR = 2.16; 95%CI 1.83-2.54) were more likely to know the transmission routes comparing with other people. Comparing with students, teachers (OR = 1.46; 95%CI 1.09-1.96) were more likely to answer the above questions Table 3 and Table 4 ). Regarding the A/H1N1vaccination, 69.9% respondents believed that the occurrence rate of adverse reactions caused by A/H1N1 vaccination was fairly low and they were not afraid of taking up vaccination. Most residents (96.1%) thought that the state's vaccination strategy was reasonable.
About half of the respondents (42.9%) had avoided going to crowded places during the past two weeks of our survey. In case people nearby held influenza-like symptoms such as fever or cough, 56.9% increased the frequency of hand-washing and 57.4% would stay away from them. Multiple logistic regression analysis indicated compliance with the preventive practices were more likely to be taken by those who were females (OR = 1. Table 3 and Table 4 ). The immunization rates of the seasonal flu and A/ H1N1 in respondents were 7.5% and 10.8% respectively. The multivariate stepwise models further showed that except the health care workers (OR = 1.52; 95%CI 1.09-2.11), residents in other occupations (OR = 0.06-0.67) were less likely to take up the A/H1N1 vaccination comparing with students (in Table 3 ). Adjusting for the background covariates the knowledge about the free vaccination policy (OR = 7.20; 95%CI 5.91-8.78) and the state's initial vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), perception of daily life disturbed (OR = 1.29; 95% CI 1.11-1.50), practice of injecting the seasonal influenza vaccine (OR = 4.69; 95%CI 3.53-6.23) were significantly associated with behavior of taking up the A/H1N1 vaccination positively (in Table 5 ), and the adverse reaction of A/H1N1 vaccine negatively influenced people's practice (OR = 0.07; 95%CI 0.04-0.11).
Novel A/H1N1 has caused pandemic in this century. It is important to encourage the public to adopt precautionary behaviors, which is based on the correct knowledge of the epidemic and appropriate response among residents. Many studies have examined the various levels of KAP about infectious disease outbreaks, such as SARS, avian influenza [13] [14] [15] . Some studies have been reported specifically on community responses to A/H1N1 in Australia and Europe [16, 17] . But through literature search, we haven't found any public reports on KAP regarding A/H1N1 among Chinese population until now. Therefore, we conducted this large population-based survey (10669 respondents) to investigate community responses to A/H1N1 and to provide baseline data to government for preventive measures in case of future outbreaks.
Unless people have basic knowledge about the modes of transmission, they respond appropriately during an outbreak [16] . It has been proved that influenza is transmitted through person to person via respiratory secretions [18] . Most residents in our survey recognized that OR m : odds ratio obtained from stepwise multivariate logistics regression analysis using univariately significant variables as candidate variables and adjusting for region; NU: not significant in the univariate analysis; *: P < 0.05; †: P < 0.01; ‡: P < 0.0001.
the risk of getting infected would increase when an infected person coughed or sneezed in close distance. This may be due to the previous experience of SARS and avian flu. Multivariate analysis results showed that workers and farmers with lower education level were less likely to have this knowledge, which indicated that the contents and forms of propaganda should be more understandable and acceptable. A large proportion of residents in our survey overlooked the indirect hand contact and hand-shaking transmission route and about one third of public misconceived that A/H1N1 was food borne, which was associated with the previous knowledge of avian flu and the new A/H1N1 flu in the general population. The confusion with avian flu might mislead some residents to believe that the A/H1N1 virus is fatal and cause public panic [19] . Therefore, it is important for the government and health authorities to provide continuously updated information of the emerging disease through televisions, newspapers, radios, and Internet. There are regional differences in the perception of A/H1N1. For example, the public in Hong Kong did not perceive a high likelihood of having a local A/H1N1 outbreak [19] , but Malaysians were particularly anxious about the pandemic [20] . The current study shows that emotional distress was relatively mild in China as few residents worried about being infected (25.1%). This phenomenon may also be related to the previous experience of the SARS epidemic, as well as the open epidemic information. A survey in Korean university showed that women perceived higher illness severity and personal susceptibility to A/ H1N1 infection, which had been reconfirmed in our study [21] . Logistic regression analysis results suggested that women with higher educational level had higher perception of risk. As time went by, the knowledge about the main transmission route increased, but the risk perception of being infected in residents decreased, suggesting the positive effect of government policy regarding A/H1N1 infection prevention, as well as the promotion of the media.
The previous study presented various results of influencing factors on the the compliance with the preventive practices. The study in Saudi showed that older men with better education were more likely to take preventive practices [9] ; female students in Korean washed hands more frequently during the peak pandemic period of A/ H1N1 [21] ; in another pandemic study in USA, younger people was found to have greater uptake of recommended behaviors but not for gender [16] . We found female with higher education took more precautionary behaviors, but office staffs and farmers took less comparing with students. While such differences could result from study population demographics, profound differences may also exist in the knowledge of A/H1N1 and the perceptions of recommended behaviors in those countries. Adjusting for the background factors, the multivariate logistic regression showed the possible relationship between knowledge and risk perception, knowledge and practices (odd ratios were 1.57 and 2.09, respectively), which indicated that good knowledge is important to enable individuals to have better attitudes and practices in influenza risk reduction. Similar findings were observed in other studies performed during A/ H1N1 pandemic in Singapore [22] and during SARS pandemic in Hong Kong [13] . Therefore, it is important to focus on inculcating the correct knowledge to individuals as it will influence both attitudes and practices. Injecting vaccination is an effective measure to prevent infectious disease [23] . In China, the seasonal influenza vaccination is not included in the national immunization program and must be purchased by recipients. Those who are above 60 years old, the pupil and children in kindergarten, and people with chronic diseases are recommended to get inoculation. Data provided by China CDC in 2009 showed that the immunization rate of the seasonal flu in Chinese population was below 2% [24] , which was much lower than 7.5% in our study (P < 0.0001). This phenomenon is partly due to the state's prior vaccination strategy for population at high risk such as students, teachers, healthcare workers and people with chronic disease, as well as the confusion between seasonal flu vaccine and A/H1N1 vaccine in residents. People who couldn't access the A/H1N1 vaccine may take up seasonal flu vaccine as preventive behaviors. The A/ H1N1 vaccine was not available in China until the middle of September 2009. All populations at high risk above three years old were invited for vaccination free of charge [25] . A survey among 868 European travelers showed 14.2% participants were vaccinated against pandemic influenza A/H1N1 [26] , higher than 10.8% in our study (P < 0.01). Our study also showed students and health care workers were more likely to take up, which may be due to the prior vaccination strategy. Multivariate stepwise logistic regression analysis, which allowed us to adjust for background factors, further showed the perceived risk of infection and the knowledge about the main modes of transmission related to A/H1N1 vaccination were insignificantly, similar results seen in Lau's study [8] . Therefore, the vaccination rate of A/H1N1 is not expected to increase even if the virus becomes more prevalent or the knowledge of its transmission mode improved. Additionally, the behavior of taking up A/H1N1 vaccine was associated with perceptions of vaccine's safety and influence on daily life by A/H1N1 as well as the knowledge about the free vaccination policy and the state's initial vaccination strategy. This suggests that improving the safety of vaccine, the acceptability of side effect and the knowledge about the state's strategy related to A/H1N1 vaccination in residents may be helpful to promote A/H1N1 vaccination in the general population. The cross-sectional telephone survey adopted in the study has some limitations. We were unable to interview the people who did not have phones and the depth of the questionnaire was largely limited because questions and pre-existing answers could not be too long and complex. In addition, the telephone response rate was 46.8%, which means more than half of the interviewees rejected or didn't finish the survey. It was impossible to compare the difference between respondents and nonrespondents due to the lack of their basic information.
This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic as most of them confused the transmission route of A/H1N1. There are many factors influencing the KAP related to A/H1N1. Female with higher educational level had higher perceived risk of infection and took more precautionary behaviors. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account. The data collected in this survey could be used as baseline data to monitor public perceives and behaviors in the event of future outbreak of infectious disease in China.
Additional file 1: Questionnaire. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China. | 2,675 | What is the highest alert level given by the World Health Organization to a pandemic? | {
"answer_start": [
2938
],
"text": [
"phase 6"
]
} | 525 |
814 | Knowledge, Attitudes and Practices (KAP) related to the Pandemic (H1N1) 2009 among Chinese General Population: a Telephone Survey
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112099/
SHA: fe954b75ed45c02d47090ee70d25c726b24b081c
Authors: Lin, Yilan; Huang, Lijuan; Nie, Shaofa; Liu, Zengyan; Yu, Hongjie; Yan, Weirong; Xu, Yihua
Date: 2011-05-16
DOI: 10.1186/1471-2334-11-128
License: cc-by
Abstract: BACKGROUND: China is at greatest risk of the Pandemic (H1N1) 2009 due to its huge population and high residential density. The unclear comprehension and negative attitudes towards the emerging infectious disease among general population may lead to unnecessary worry and even panic. The objective of this study was to investigate the Chinese public response to H1N1 pandemic and provide baseline data to develop public education campaigns in response to future outbreaks. METHODS: A close-ended questionnaire developed by the Chinese Center for Disease Control and Prevention was applied to assess the knowledge, attitudes and practices (KAP) of pandemic (H1N1) 2009 among 10,669 responders recruited from seven urban and two rural areas of China sampled by using the probability proportional to size (PPS) method. RESULTS: 30.0% respondents were not clear whether food spread H1N1 virusand. 65.7% reported that the pandemic had no impact on their life. The immunization rates of the seasonal flu and H1N1vaccine were 7.5% and 10.8%, respectively. Farmers and those with lower education level were less likely to know the main transmission route (cough or talk face to face). Female and those with college and above education had higher perception of risk and more compliance with preventive behaviors. Relationships between knowledge and risk perception (OR = 1.69; 95%CI 1.54-1.86), and knowledge and practices (OR = 1.57; 95%CI 1.42-1.73) were found among the study subjects. With regard to the behavior of taking up A/H1N1 vaccination, there are several related factors found in the current study population, including the perception of life disturbed (OR = 1.29; 95%CI 1.11-1.50), the safety of A/H1N1 vaccine (OR = 0.07; 95%CI 0.04-0.11), the knowledge of free vaccination policy (OR = 7.20; 95%CI 5.91-8.78), the state's priority vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), and taking up seasonal influenza vaccine behavior (OR = 4.69; 95%CI 3.53-6.23). CONCLUSIONS: This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account.
Text: At the end of March 2009, an outbreak of novel influenza A (H1N1) (here after called A/H1N1) infection occurred in Mexico, followed by ongoing spread to all over the world in a short period [1] . On June 11 2009, the World Health Organization raised its pandemic alert level to the highest level, phase 6 [2] , meaning that the A/H1N1 flu had spread in more than two continents and reached pandemic proportions. As of June 13, 2010, it had caused over 18,172 deaths in more than 214 countries and overseas territories or communities [3] . Most illness, especially the severe illness and deaths, had occurred among healthy young adults, which was markedly different from the disease pattern seen during epidemics of seasonal influenza [4, 5] .
China is highly susceptible to A/H1N1 because of its huge population and high residential density, besides the high infectiousness of this novel influenza virus. After the first imported case reported on May 11, 2009 , the confirmed cases were reported in various provinces of China [6] . By the late of October 2009, A/H1N1 cases had increased dramatically, with 44,981 cases and 6 deaths confirmed at the end of October 2009. The A/ H1N1 infection rate peaked in November 2009, when approximately 1500 new cases of A/H1N1 were being confirmed each day. By the end of this month, a total of 92,904 cases and 200 deaths had resulted from A/ H1N1-related causes [7] . The Chinese government has taken a series of preventive measures according to WHO guidelines, including the promotion of public knowledge about flu through mass media, patient isolation, quarantine of close contact person, and free vaccinations to population at high risk (e.g. young children, healthcare workers, and people with chronic disease) [8] . However, there were few public reports on the assessment of the effect of these policies and the level of knowledge, attitude and practice (KAP) associating with A/H1N1 among general population.
It is well-known that confused comprehension and negative attitude towards the emerging communicable disease may lead to unnecessary worry and chaos, even excessive panic which would aggravate the disease epidemic [9] . For instance, during SARS epidemic from 2002 to 2004, the misconceptions and the excessive panic of Chinese public to SARS led the public resistant to comply with the suggested preventive measures such as avoiding public transportation, going to hospital when they were sick, which contributed to the rapid spread of SARS and resulted in a more serious epidemic situation, making China one of the worst affected countries with over 5327 cases and 439 deaths [10, 11] . In addition, the panic of infectious disease outbreak could cause huge economic loss, for example the economic loss of SARS has been estimated at $30-$100 billion in US, though less than 10,000 persons were infected [12] . SARS experience has demonstrated the importance of monitoring the public perception in disease epidemic control, which may affect the compliance of community to the precautionary strategies. Understanding related factors affecting people to undertake precautionary behavior may also help decision-makers take appropriate measures to promote individual or community health. Therefore, it is important to monitor and analyze the public response to the emerging disease.
To investigate community responses to A/H1N1 in China, we conducted this telephone survey to describe the knowledge, attitudes and practices of A/H1N1 among general population in China and put forward policy recommendations to government in case of future similar conditions.
This study was performed in seven urban regions (Beijing, Shanghai, Wuhan, Jingzhou, Xi'an, Zhengzhou, Shenzhen cities) and two rural areas (Jingzhou and Zhengzhou counties) of China with over one million people in each region. Regarding the urban sites, Beijing as the capital of China locates in the northeast; Shanghai is a municipality in the east of China; Wuhan (the provincial capital of Hubei) and Zhengzhou (the provincial capital of Henan province) are both in the centre of China; Xi'an in the northwest of China is the provincial capital of Shanxi province; and Shenzhen of the Guangdong province is in the southeast of China. As for the rural sites, Jingzhou county and Zhengzhou county, from Hubei and Henan provinces, respectively, both locate in the centre of China.
This current study was carried out in three phases during the pandemic peak season of A/H1N1. The first phase was from 30 November 2009 to 27 December 2009, the second from 4 January 2010 to 24 January 2010, and the third from 24 February to 25 March in 2010.
A two-stage proportional probability to size (PPS) sampling method was used in each phase. In stage І, about 30% of administrative regions in each study site were selected as primary sample units (PSUs) for cluster sampling. In stage II, telephone numbers were sampled randomly, of which the first four digitals were obtained from each PSU's post office as initial number and the other three or four digitals were obtained from random number generated by Excel 2003. Then each family was chosen as per unit (excluding school, hotel public or cell phone etc.) and at least 400 families in each site at each phase were selected finally. If the family was selected repeatedly or refused to answer the questionnaire, we added one to the last digit of phone number and dial again. If the line was busy or of no response, we would dial three times and then give up this phone number if there was still no respondent.
Anonymous telephone interviews were conducted from 6:30 pm to 10:00 pm so as to avoid over-presenting the non-work population by well-trained interviewers with Bachelor degree of Epidemiology. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of A/H1N1 Pandemic by Telephone was designed by the Chinese Centre for Disease Control and Prevention (China CDC, Beijing). The majority of the questions were closed-ended and variables in the questionnaire were categorical, except age. The inclusion criteria of subjects were: age≥18 and proper communication skills. There were seven questions related to the knowledge of A/H1N1, four referred to the attitude, and five concerning about the practice in this questionnaire (See additional file 1: The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China).
This study was approved by the institutional review board of the Tongji Medical College of Huazhong University of Science and Technology. All respondents were informed consent. We respected their wishes whether to accept our survey and promised to protect their secrets.
All data were entered into computer using Epidata V.3.1 and were analyzed in SPSS statistical software V.12. Chi-square test was applied to compare the immunization rates of the seasonal flu and A/H1N1 vaccine. The associations between the socio-demographic factors and the KAP regarding A/H1N1 were firstly investigated by using univariate odds ratios (OR) and then stepwise logistic regression modeling applied. Adjusting for such background variables including gender, age, level of education, occupation, region, and survey wave, stepwise multivariate logistic regression models were applied to investigate the impact factors associated with the risk perception of A/H1N1, A/H1N1 vaccination uptake and the compliance with suggested preventive measures (avoid crowd places/wash hand frequently/keep distance from people with influenza-like symptoms). For the purposes of analysis, the factor knowledge about the main modes of transmission was divided into two groups according to whether the respondents knew both cough and talk faceto-face can spread A/H1N1. Odds ratios and respective 95% confidence intervals (CI) were obtained from the logistic regression analysis. P values lower than 0.05 were judged to be statistically significant.
A total of 88541 telephone numbers were dialed. Except 65323 invalid calls (including vacant numbers, fax numbers, busy tone numbers and non-qualified respondents whose age <18 and whose phones were from school, hotel or other public places), 23218 eligible respondents were identified. Among these respondents, 12360 completed the interview. Therefore, the response rate was 46.8%. Excluding missing, and logical erroneous data, 10669 questionnaires in total were eligible for analysis. The baseline characteristics of the respondents were presented in Table1. The mean age of all respondents was 41.47 years (over range: 18-90 year) . Of all respondents, 54.4% were female, and 42.4% had received college or above education (Table 1) .
The overall KAP related to A/H1N1 was reported in Table 2 . As to knowledge, 75.6% of all respondents knew that influenza could be transmitted by coughing and sneezing, and 61.9% thought that talking face-to-face was the transmission route, whereas 30.0% believed the transmission could be through food. Less than one third of respondents knew that virus could be transmitted by handshaking and indirect hand contact (26.8% and 22.3%, respectively). Multiple logistic regression analysis showed that those with middle school (OR = 1.71; 95%CI 1.48-1.98), or having an education level of college and above (OR = 2.16; 95%CI 1.83-2.54) were more likely to know the transmission routes comparing with other people. Comparing with students, teachers (OR = 1.46; 95%CI 1.09-1.96) were more likely to answer the above questions Table 3 and Table 4 ). Regarding the A/H1N1vaccination, 69.9% respondents believed that the occurrence rate of adverse reactions caused by A/H1N1 vaccination was fairly low and they were not afraid of taking up vaccination. Most residents (96.1%) thought that the state's vaccination strategy was reasonable.
About half of the respondents (42.9%) had avoided going to crowded places during the past two weeks of our survey. In case people nearby held influenza-like symptoms such as fever or cough, 56.9% increased the frequency of hand-washing and 57.4% would stay away from them. Multiple logistic regression analysis indicated compliance with the preventive practices were more likely to be taken by those who were females (OR = 1. Table 3 and Table 4 ). The immunization rates of the seasonal flu and A/ H1N1 in respondents were 7.5% and 10.8% respectively. The multivariate stepwise models further showed that except the health care workers (OR = 1.52; 95%CI 1.09-2.11), residents in other occupations (OR = 0.06-0.67) were less likely to take up the A/H1N1 vaccination comparing with students (in Table 3 ). Adjusting for the background covariates the knowledge about the free vaccination policy (OR = 7.20; 95%CI 5.91-8.78) and the state's initial vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), perception of daily life disturbed (OR = 1.29; 95% CI 1.11-1.50), practice of injecting the seasonal influenza vaccine (OR = 4.69; 95%CI 3.53-6.23) were significantly associated with behavior of taking up the A/H1N1 vaccination positively (in Table 5 ), and the adverse reaction of A/H1N1 vaccine negatively influenced people's practice (OR = 0.07; 95%CI 0.04-0.11).
Novel A/H1N1 has caused pandemic in this century. It is important to encourage the public to adopt precautionary behaviors, which is based on the correct knowledge of the epidemic and appropriate response among residents. Many studies have examined the various levels of KAP about infectious disease outbreaks, such as SARS, avian influenza [13] [14] [15] . Some studies have been reported specifically on community responses to A/H1N1 in Australia and Europe [16, 17] . But through literature search, we haven't found any public reports on KAP regarding A/H1N1 among Chinese population until now. Therefore, we conducted this large population-based survey (10669 respondents) to investigate community responses to A/H1N1 and to provide baseline data to government for preventive measures in case of future outbreaks.
Unless people have basic knowledge about the modes of transmission, they respond appropriately during an outbreak [16] . It has been proved that influenza is transmitted through person to person via respiratory secretions [18] . Most residents in our survey recognized that OR m : odds ratio obtained from stepwise multivariate logistics regression analysis using univariately significant variables as candidate variables and adjusting for region; NU: not significant in the univariate analysis; *: P < 0.05; †: P < 0.01; ‡: P < 0.0001.
the risk of getting infected would increase when an infected person coughed or sneezed in close distance. This may be due to the previous experience of SARS and avian flu. Multivariate analysis results showed that workers and farmers with lower education level were less likely to have this knowledge, which indicated that the contents and forms of propaganda should be more understandable and acceptable. A large proportion of residents in our survey overlooked the indirect hand contact and hand-shaking transmission route and about one third of public misconceived that A/H1N1 was food borne, which was associated with the previous knowledge of avian flu and the new A/H1N1 flu in the general population. The confusion with avian flu might mislead some residents to believe that the A/H1N1 virus is fatal and cause public panic [19] . Therefore, it is important for the government and health authorities to provide continuously updated information of the emerging disease through televisions, newspapers, radios, and Internet. There are regional differences in the perception of A/H1N1. For example, the public in Hong Kong did not perceive a high likelihood of having a local A/H1N1 outbreak [19] , but Malaysians were particularly anxious about the pandemic [20] . The current study shows that emotional distress was relatively mild in China as few residents worried about being infected (25.1%). This phenomenon may also be related to the previous experience of the SARS epidemic, as well as the open epidemic information. A survey in Korean university showed that women perceived higher illness severity and personal susceptibility to A/ H1N1 infection, which had been reconfirmed in our study [21] . Logistic regression analysis results suggested that women with higher educational level had higher perception of risk. As time went by, the knowledge about the main transmission route increased, but the risk perception of being infected in residents decreased, suggesting the positive effect of government policy regarding A/H1N1 infection prevention, as well as the promotion of the media.
The previous study presented various results of influencing factors on the the compliance with the preventive practices. The study in Saudi showed that older men with better education were more likely to take preventive practices [9] ; female students in Korean washed hands more frequently during the peak pandemic period of A/ H1N1 [21] ; in another pandemic study in USA, younger people was found to have greater uptake of recommended behaviors but not for gender [16] . We found female with higher education took more precautionary behaviors, but office staffs and farmers took less comparing with students. While such differences could result from study population demographics, profound differences may also exist in the knowledge of A/H1N1 and the perceptions of recommended behaviors in those countries. Adjusting for the background factors, the multivariate logistic regression showed the possible relationship between knowledge and risk perception, knowledge and practices (odd ratios were 1.57 and 2.09, respectively), which indicated that good knowledge is important to enable individuals to have better attitudes and practices in influenza risk reduction. Similar findings were observed in other studies performed during A/ H1N1 pandemic in Singapore [22] and during SARS pandemic in Hong Kong [13] . Therefore, it is important to focus on inculcating the correct knowledge to individuals as it will influence both attitudes and practices. Injecting vaccination is an effective measure to prevent infectious disease [23] . In China, the seasonal influenza vaccination is not included in the national immunization program and must be purchased by recipients. Those who are above 60 years old, the pupil and children in kindergarten, and people with chronic diseases are recommended to get inoculation. Data provided by China CDC in 2009 showed that the immunization rate of the seasonal flu in Chinese population was below 2% [24] , which was much lower than 7.5% in our study (P < 0.0001). This phenomenon is partly due to the state's prior vaccination strategy for population at high risk such as students, teachers, healthcare workers and people with chronic disease, as well as the confusion between seasonal flu vaccine and A/H1N1 vaccine in residents. People who couldn't access the A/H1N1 vaccine may take up seasonal flu vaccine as preventive behaviors. The A/ H1N1 vaccine was not available in China until the middle of September 2009. All populations at high risk above three years old were invited for vaccination free of charge [25] . A survey among 868 European travelers showed 14.2% participants were vaccinated against pandemic influenza A/H1N1 [26] , higher than 10.8% in our study (P < 0.01). Our study also showed students and health care workers were more likely to take up, which may be due to the prior vaccination strategy. Multivariate stepwise logistic regression analysis, which allowed us to adjust for background factors, further showed the perceived risk of infection and the knowledge about the main modes of transmission related to A/H1N1 vaccination were insignificantly, similar results seen in Lau's study [8] . Therefore, the vaccination rate of A/H1N1 is not expected to increase even if the virus becomes more prevalent or the knowledge of its transmission mode improved. Additionally, the behavior of taking up A/H1N1 vaccine was associated with perceptions of vaccine's safety and influence on daily life by A/H1N1 as well as the knowledge about the free vaccination policy and the state's initial vaccination strategy. This suggests that improving the safety of vaccine, the acceptability of side effect and the knowledge about the state's strategy related to A/H1N1 vaccination in residents may be helpful to promote A/H1N1 vaccination in the general population. The cross-sectional telephone survey adopted in the study has some limitations. We were unable to interview the people who did not have phones and the depth of the questionnaire was largely limited because questions and pre-existing answers could not be too long and complex. In addition, the telephone response rate was 46.8%, which means more than half of the interviewees rejected or didn't finish the survey. It was impossible to compare the difference between respondents and nonrespondents due to the lack of their basic information.
This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic as most of them confused the transmission route of A/H1N1. There are many factors influencing the KAP related to A/H1N1. Female with higher educational level had higher perceived risk of infection and took more precautionary behaviors. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account. The data collected in this survey could be used as baseline data to monitor public perceives and behaviors in the event of future outbreak of infectious disease in China.
Additional file 1: Questionnaire. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China. | 2,675 | What does it mean for a pandemic to have a WHO alert level of 6? | {
"answer_start": [
2984
],
"text": [
"spread in more than two continents"
]
} | 527 |
815 | Knowledge, Attitudes and Practices (KAP) related to the Pandemic (H1N1) 2009 among Chinese General Population: a Telephone Survey
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112099/
SHA: fe954b75ed45c02d47090ee70d25c726b24b081c
Authors: Lin, Yilan; Huang, Lijuan; Nie, Shaofa; Liu, Zengyan; Yu, Hongjie; Yan, Weirong; Xu, Yihua
Date: 2011-05-16
DOI: 10.1186/1471-2334-11-128
License: cc-by
Abstract: BACKGROUND: China is at greatest risk of the Pandemic (H1N1) 2009 due to its huge population and high residential density. The unclear comprehension and negative attitudes towards the emerging infectious disease among general population may lead to unnecessary worry and even panic. The objective of this study was to investigate the Chinese public response to H1N1 pandemic and provide baseline data to develop public education campaigns in response to future outbreaks. METHODS: A close-ended questionnaire developed by the Chinese Center for Disease Control and Prevention was applied to assess the knowledge, attitudes and practices (KAP) of pandemic (H1N1) 2009 among 10,669 responders recruited from seven urban and two rural areas of China sampled by using the probability proportional to size (PPS) method. RESULTS: 30.0% respondents were not clear whether food spread H1N1 virusand. 65.7% reported that the pandemic had no impact on their life. The immunization rates of the seasonal flu and H1N1vaccine were 7.5% and 10.8%, respectively. Farmers and those with lower education level were less likely to know the main transmission route (cough or talk face to face). Female and those with college and above education had higher perception of risk and more compliance with preventive behaviors. Relationships between knowledge and risk perception (OR = 1.69; 95%CI 1.54-1.86), and knowledge and practices (OR = 1.57; 95%CI 1.42-1.73) were found among the study subjects. With regard to the behavior of taking up A/H1N1 vaccination, there are several related factors found in the current study population, including the perception of life disturbed (OR = 1.29; 95%CI 1.11-1.50), the safety of A/H1N1 vaccine (OR = 0.07; 95%CI 0.04-0.11), the knowledge of free vaccination policy (OR = 7.20; 95%CI 5.91-8.78), the state's priority vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), and taking up seasonal influenza vaccine behavior (OR = 4.69; 95%CI 3.53-6.23). CONCLUSIONS: This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account.
Text: At the end of March 2009, an outbreak of novel influenza A (H1N1) (here after called A/H1N1) infection occurred in Mexico, followed by ongoing spread to all over the world in a short period [1] . On June 11 2009, the World Health Organization raised its pandemic alert level to the highest level, phase 6 [2] , meaning that the A/H1N1 flu had spread in more than two continents and reached pandemic proportions. As of June 13, 2010, it had caused over 18,172 deaths in more than 214 countries and overseas territories or communities [3] . Most illness, especially the severe illness and deaths, had occurred among healthy young adults, which was markedly different from the disease pattern seen during epidemics of seasonal influenza [4, 5] .
China is highly susceptible to A/H1N1 because of its huge population and high residential density, besides the high infectiousness of this novel influenza virus. After the first imported case reported on May 11, 2009 , the confirmed cases were reported in various provinces of China [6] . By the late of October 2009, A/H1N1 cases had increased dramatically, with 44,981 cases and 6 deaths confirmed at the end of October 2009. The A/ H1N1 infection rate peaked in November 2009, when approximately 1500 new cases of A/H1N1 were being confirmed each day. By the end of this month, a total of 92,904 cases and 200 deaths had resulted from A/ H1N1-related causes [7] . The Chinese government has taken a series of preventive measures according to WHO guidelines, including the promotion of public knowledge about flu through mass media, patient isolation, quarantine of close contact person, and free vaccinations to population at high risk (e.g. young children, healthcare workers, and people with chronic disease) [8] . However, there were few public reports on the assessment of the effect of these policies and the level of knowledge, attitude and practice (KAP) associating with A/H1N1 among general population.
It is well-known that confused comprehension and negative attitude towards the emerging communicable disease may lead to unnecessary worry and chaos, even excessive panic which would aggravate the disease epidemic [9] . For instance, during SARS epidemic from 2002 to 2004, the misconceptions and the excessive panic of Chinese public to SARS led the public resistant to comply with the suggested preventive measures such as avoiding public transportation, going to hospital when they were sick, which contributed to the rapid spread of SARS and resulted in a more serious epidemic situation, making China one of the worst affected countries with over 5327 cases and 439 deaths [10, 11] . In addition, the panic of infectious disease outbreak could cause huge economic loss, for example the economic loss of SARS has been estimated at $30-$100 billion in US, though less than 10,000 persons were infected [12] . SARS experience has demonstrated the importance of monitoring the public perception in disease epidemic control, which may affect the compliance of community to the precautionary strategies. Understanding related factors affecting people to undertake precautionary behavior may also help decision-makers take appropriate measures to promote individual or community health. Therefore, it is important to monitor and analyze the public response to the emerging disease.
To investigate community responses to A/H1N1 in China, we conducted this telephone survey to describe the knowledge, attitudes and practices of A/H1N1 among general population in China and put forward policy recommendations to government in case of future similar conditions.
This study was performed in seven urban regions (Beijing, Shanghai, Wuhan, Jingzhou, Xi'an, Zhengzhou, Shenzhen cities) and two rural areas (Jingzhou and Zhengzhou counties) of China with over one million people in each region. Regarding the urban sites, Beijing as the capital of China locates in the northeast; Shanghai is a municipality in the east of China; Wuhan (the provincial capital of Hubei) and Zhengzhou (the provincial capital of Henan province) are both in the centre of China; Xi'an in the northwest of China is the provincial capital of Shanxi province; and Shenzhen of the Guangdong province is in the southeast of China. As for the rural sites, Jingzhou county and Zhengzhou county, from Hubei and Henan provinces, respectively, both locate in the centre of China.
This current study was carried out in three phases during the pandemic peak season of A/H1N1. The first phase was from 30 November 2009 to 27 December 2009, the second from 4 January 2010 to 24 January 2010, and the third from 24 February to 25 March in 2010.
A two-stage proportional probability to size (PPS) sampling method was used in each phase. In stage І, about 30% of administrative regions in each study site were selected as primary sample units (PSUs) for cluster sampling. In stage II, telephone numbers were sampled randomly, of which the first four digitals were obtained from each PSU's post office as initial number and the other three or four digitals were obtained from random number generated by Excel 2003. Then each family was chosen as per unit (excluding school, hotel public or cell phone etc.) and at least 400 families in each site at each phase were selected finally. If the family was selected repeatedly or refused to answer the questionnaire, we added one to the last digit of phone number and dial again. If the line was busy or of no response, we would dial three times and then give up this phone number if there was still no respondent.
Anonymous telephone interviews were conducted from 6:30 pm to 10:00 pm so as to avoid over-presenting the non-work population by well-trained interviewers with Bachelor degree of Epidemiology. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of A/H1N1 Pandemic by Telephone was designed by the Chinese Centre for Disease Control and Prevention (China CDC, Beijing). The majority of the questions were closed-ended and variables in the questionnaire were categorical, except age. The inclusion criteria of subjects were: age≥18 and proper communication skills. There were seven questions related to the knowledge of A/H1N1, four referred to the attitude, and five concerning about the practice in this questionnaire (See additional file 1: The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China).
This study was approved by the institutional review board of the Tongji Medical College of Huazhong University of Science and Technology. All respondents were informed consent. We respected their wishes whether to accept our survey and promised to protect their secrets.
All data were entered into computer using Epidata V.3.1 and were analyzed in SPSS statistical software V.12. Chi-square test was applied to compare the immunization rates of the seasonal flu and A/H1N1 vaccine. The associations between the socio-demographic factors and the KAP regarding A/H1N1 were firstly investigated by using univariate odds ratios (OR) and then stepwise logistic regression modeling applied. Adjusting for such background variables including gender, age, level of education, occupation, region, and survey wave, stepwise multivariate logistic regression models were applied to investigate the impact factors associated with the risk perception of A/H1N1, A/H1N1 vaccination uptake and the compliance with suggested preventive measures (avoid crowd places/wash hand frequently/keep distance from people with influenza-like symptoms). For the purposes of analysis, the factor knowledge about the main modes of transmission was divided into two groups according to whether the respondents knew both cough and talk faceto-face can spread A/H1N1. Odds ratios and respective 95% confidence intervals (CI) were obtained from the logistic regression analysis. P values lower than 0.05 were judged to be statistically significant.
A total of 88541 telephone numbers were dialed. Except 65323 invalid calls (including vacant numbers, fax numbers, busy tone numbers and non-qualified respondents whose age <18 and whose phones were from school, hotel or other public places), 23218 eligible respondents were identified. Among these respondents, 12360 completed the interview. Therefore, the response rate was 46.8%. Excluding missing, and logical erroneous data, 10669 questionnaires in total were eligible for analysis. The baseline characteristics of the respondents were presented in Table1. The mean age of all respondents was 41.47 years (over range: 18-90 year) . Of all respondents, 54.4% were female, and 42.4% had received college or above education (Table 1) .
The overall KAP related to A/H1N1 was reported in Table 2 . As to knowledge, 75.6% of all respondents knew that influenza could be transmitted by coughing and sneezing, and 61.9% thought that talking face-to-face was the transmission route, whereas 30.0% believed the transmission could be through food. Less than one third of respondents knew that virus could be transmitted by handshaking and indirect hand contact (26.8% and 22.3%, respectively). Multiple logistic regression analysis showed that those with middle school (OR = 1.71; 95%CI 1.48-1.98), or having an education level of college and above (OR = 2.16; 95%CI 1.83-2.54) were more likely to know the transmission routes comparing with other people. Comparing with students, teachers (OR = 1.46; 95%CI 1.09-1.96) were more likely to answer the above questions Table 3 and Table 4 ). Regarding the A/H1N1vaccination, 69.9% respondents believed that the occurrence rate of adverse reactions caused by A/H1N1 vaccination was fairly low and they were not afraid of taking up vaccination. Most residents (96.1%) thought that the state's vaccination strategy was reasonable.
About half of the respondents (42.9%) had avoided going to crowded places during the past two weeks of our survey. In case people nearby held influenza-like symptoms such as fever or cough, 56.9% increased the frequency of hand-washing and 57.4% would stay away from them. Multiple logistic regression analysis indicated compliance with the preventive practices were more likely to be taken by those who were females (OR = 1. Table 3 and Table 4 ). The immunization rates of the seasonal flu and A/ H1N1 in respondents were 7.5% and 10.8% respectively. The multivariate stepwise models further showed that except the health care workers (OR = 1.52; 95%CI 1.09-2.11), residents in other occupations (OR = 0.06-0.67) were less likely to take up the A/H1N1 vaccination comparing with students (in Table 3 ). Adjusting for the background covariates the knowledge about the free vaccination policy (OR = 7.20; 95%CI 5.91-8.78) and the state's initial vaccination strategy(OR = 1.33; 95%CI 1.08-1.64), perception of daily life disturbed (OR = 1.29; 95% CI 1.11-1.50), practice of injecting the seasonal influenza vaccine (OR = 4.69; 95%CI 3.53-6.23) were significantly associated with behavior of taking up the A/H1N1 vaccination positively (in Table 5 ), and the adverse reaction of A/H1N1 vaccine negatively influenced people's practice (OR = 0.07; 95%CI 0.04-0.11).
Novel A/H1N1 has caused pandemic in this century. It is important to encourage the public to adopt precautionary behaviors, which is based on the correct knowledge of the epidemic and appropriate response among residents. Many studies have examined the various levels of KAP about infectious disease outbreaks, such as SARS, avian influenza [13] [14] [15] . Some studies have been reported specifically on community responses to A/H1N1 in Australia and Europe [16, 17] . But through literature search, we haven't found any public reports on KAP regarding A/H1N1 among Chinese population until now. Therefore, we conducted this large population-based survey (10669 respondents) to investigate community responses to A/H1N1 and to provide baseline data to government for preventive measures in case of future outbreaks.
Unless people have basic knowledge about the modes of transmission, they respond appropriately during an outbreak [16] . It has been proved that influenza is transmitted through person to person via respiratory secretions [18] . Most residents in our survey recognized that OR m : odds ratio obtained from stepwise multivariate logistics regression analysis using univariately significant variables as candidate variables and adjusting for region; NU: not significant in the univariate analysis; *: P < 0.05; †: P < 0.01; ‡: P < 0.0001.
the risk of getting infected would increase when an infected person coughed or sneezed in close distance. This may be due to the previous experience of SARS and avian flu. Multivariate analysis results showed that workers and farmers with lower education level were less likely to have this knowledge, which indicated that the contents and forms of propaganda should be more understandable and acceptable. A large proportion of residents in our survey overlooked the indirect hand contact and hand-shaking transmission route and about one third of public misconceived that A/H1N1 was food borne, which was associated with the previous knowledge of avian flu and the new A/H1N1 flu in the general population. The confusion with avian flu might mislead some residents to believe that the A/H1N1 virus is fatal and cause public panic [19] . Therefore, it is important for the government and health authorities to provide continuously updated information of the emerging disease through televisions, newspapers, radios, and Internet. There are regional differences in the perception of A/H1N1. For example, the public in Hong Kong did not perceive a high likelihood of having a local A/H1N1 outbreak [19] , but Malaysians were particularly anxious about the pandemic [20] . The current study shows that emotional distress was relatively mild in China as few residents worried about being infected (25.1%). This phenomenon may also be related to the previous experience of the SARS epidemic, as well as the open epidemic information. A survey in Korean university showed that women perceived higher illness severity and personal susceptibility to A/ H1N1 infection, which had been reconfirmed in our study [21] . Logistic regression analysis results suggested that women with higher educational level had higher perception of risk. As time went by, the knowledge about the main transmission route increased, but the risk perception of being infected in residents decreased, suggesting the positive effect of government policy regarding A/H1N1 infection prevention, as well as the promotion of the media.
The previous study presented various results of influencing factors on the the compliance with the preventive practices. The study in Saudi showed that older men with better education were more likely to take preventive practices [9] ; female students in Korean washed hands more frequently during the peak pandemic period of A/ H1N1 [21] ; in another pandemic study in USA, younger people was found to have greater uptake of recommended behaviors but not for gender [16] . We found female with higher education took more precautionary behaviors, but office staffs and farmers took less comparing with students. While such differences could result from study population demographics, profound differences may also exist in the knowledge of A/H1N1 and the perceptions of recommended behaviors in those countries. Adjusting for the background factors, the multivariate logistic regression showed the possible relationship between knowledge and risk perception, knowledge and practices (odd ratios were 1.57 and 2.09, respectively), which indicated that good knowledge is important to enable individuals to have better attitudes and practices in influenza risk reduction. Similar findings were observed in other studies performed during A/ H1N1 pandemic in Singapore [22] and during SARS pandemic in Hong Kong [13] . Therefore, it is important to focus on inculcating the correct knowledge to individuals as it will influence both attitudes and practices. Injecting vaccination is an effective measure to prevent infectious disease [23] . In China, the seasonal influenza vaccination is not included in the national immunization program and must be purchased by recipients. Those who are above 60 years old, the pupil and children in kindergarten, and people with chronic diseases are recommended to get inoculation. Data provided by China CDC in 2009 showed that the immunization rate of the seasonal flu in Chinese population was below 2% [24] , which was much lower than 7.5% in our study (P < 0.0001). This phenomenon is partly due to the state's prior vaccination strategy for population at high risk such as students, teachers, healthcare workers and people with chronic disease, as well as the confusion between seasonal flu vaccine and A/H1N1 vaccine in residents. People who couldn't access the A/H1N1 vaccine may take up seasonal flu vaccine as preventive behaviors. The A/ H1N1 vaccine was not available in China until the middle of September 2009. All populations at high risk above three years old were invited for vaccination free of charge [25] . A survey among 868 European travelers showed 14.2% participants were vaccinated against pandemic influenza A/H1N1 [26] , higher than 10.8% in our study (P < 0.01). Our study also showed students and health care workers were more likely to take up, which may be due to the prior vaccination strategy. Multivariate stepwise logistic regression analysis, which allowed us to adjust for background factors, further showed the perceived risk of infection and the knowledge about the main modes of transmission related to A/H1N1 vaccination were insignificantly, similar results seen in Lau's study [8] . Therefore, the vaccination rate of A/H1N1 is not expected to increase even if the virus becomes more prevalent or the knowledge of its transmission mode improved. Additionally, the behavior of taking up A/H1N1 vaccine was associated with perceptions of vaccine's safety and influence on daily life by A/H1N1 as well as the knowledge about the free vaccination policy and the state's initial vaccination strategy. This suggests that improving the safety of vaccine, the acceptability of side effect and the knowledge about the state's strategy related to A/H1N1 vaccination in residents may be helpful to promote A/H1N1 vaccination in the general population. The cross-sectional telephone survey adopted in the study has some limitations. We were unable to interview the people who did not have phones and the depth of the questionnaire was largely limited because questions and pre-existing answers could not be too long and complex. In addition, the telephone response rate was 46.8%, which means more than half of the interviewees rejected or didn't finish the survey. It was impossible to compare the difference between respondents and nonrespondents due to the lack of their basic information.
This A/H1N1 epidemic has not caused public panic yet, but the knowledge of A/H1N1 in residents is not optimistic as most of them confused the transmission route of A/H1N1. There are many factors influencing the KAP related to A/H1N1. Female with higher educational level had higher perceived risk of infection and took more precautionary behaviors. Public education campaign may take the side effects of vaccine and the knowledge about the state's vaccination strategy into account. The data collected in this survey could be used as baseline data to monitor public perceives and behaviors in the event of future outbreak of infectious disease in China.
Additional file 1: Questionnaire. The Questionnaire to Survey the Level of Knowledge, Attitude and Practice in Different Stages of H1N1 Pandemic by Telephone in China. | 2,675 | What was the estimated economic impact in the U.S. from the 2009 SARS pandemic? | {
"answer_start": [
5423
],
"text": [
"estimated at $30-$100 billion"
]
} | 528 |
816 | The Extent of Transmission of Novel Coronavirus in Wuhan, China, 2020
https://doi.org/10.3390/jcm9020330
SHA: 919c524f19f79213e6f81aa38502c70287d273dc
Authors: Nishiura, Hiroshi; Jung, Sung-mok; Linton, Natalie M.; Kinoshita, Ryo; Yang, Yichi; Hayashi, Katsuma; Kobayashi, Tetsuro; Yuan, Baoyin; Akhmetzhanov, Andrei R.
Date: 2020
DOI: 10.3390/jcm9020330
License: cc-by
Abstract: A cluster of pneumonia cases linked to a novel coronavirus (2019-nCoV) was reported by China in late December 2019. Reported case incidence has now reached the hundreds, but this is likely an underestimate. As of 24 January 2020, with reports of thirteen exportation events, we estimate the cumulative incidence in China at 5502 cases (95% confidence interval: 3027, 9057). The most plausible number of infections is in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than the one in the epidemiologically linked seafood market in Wuhan have occurred.
Text: Since the announcement of a cluster of pneumonia cases of unknown etiology in Wuhan, Hubei Province, China, was made on 31 December 2019, many rapid virological, clinical, and epidemiological research responses have taken place [1, 2] . The causative agent of the pneumonia is suggested to be a novel coronavirus (2019-nCoV) of the same lineage (but genetically distinct) from the coronavirus causing severe acute respiratory syndrome (SARS) [1] . Cases in the initial cluster reported a common exposure-a seafood market in Wuhan where wild animals were served at a restaurant-indicating that a point-source zoonotic (animal-to-human) route was likely the main mode of transmission for those cases [2] .
Although early reports from Wuhan [3] stated that (i) there were only tens of cases in the cluster and (ii) no human-to-human transmission was directly observed, the scientific community was alert to the possibility that the novel coronavirus would spread to other geographic locations-including other countries-via direct human-to-human transmission. In early January, the outbreak began to escalate rapidly with hundreds of cases now confirmed along with the presence of a few household clusters [4] [5] [6] [7] .
As of 24 January 2020, the cumulative incidence in China is 830 cases, of which 549 cases were diagnosed in Hubei, 26 in Beijing, 20 in Shanghai, and 53 in Guangdong. Additionally, twenty-six deaths have been linked to the outbreak [6, 8] , and thirteen cases were exported to Japan, Singapore, South Korea, Taiwan, Thailand, Vietnam and the United States as of 22 January 2020. Considering that enhanced surveillance has been underway in these importing countries, case ascertainment has been perhaps better in exported case data.
Using a spatial back-calculation method and analyzing exported cases, we estimate the cumulative incidence of 2019-nCoV cases in China in real time, allowing us to update and discuss the extent of transmission at the source. Table 1 shows the incidence of exported cases by date of hospitalization and report. Due to the initial difficulty of diagnosis in the absence of established primer for polymerase chain reaction testing, the time lag between hospitalization and reporting was longer for early cases compared with that of more recent cases. Among the seven locations reporting importation, the total volume of inbound passengers from China was m = 63.1 million per year in 2017 [9] , of which 100q = 2.1% were from Wuhan [10] , a home of n = 19.0 million people as the catchment population of Wuhan airport. Two other locations with confirmed cases, i.e., Macau and Hong Kong, were excluded from the analysis, because it is commutable by land transporation and the first case in Hong Kong was indeed not via airtravel. As we already know from elsewhere [11] [12] [13] , given the observed cumulative count of c exported cases, we have a balance equation of the cumulative risk of infection:
where T is the sum of incubation and infectious periods, and here is assumed to be 3.2 and 9.3 days [14] , respectively, assuming that these periods are similar to those of other coronaviruses, and thus, T = 12.5 days. The estimated incidence in China is then given bypn. With an ad-hoc assumption that the data are generated following the binomial sampling process among travelers from Wuhan, the cumulative incidence is then estimated using a maximum likelihood method. Table 1 also shows the estimated incidence in China. The first exportation event in Thailand suggests 423 cases with the upper confidence limit of 1863 cases. The estimated cumulative incidence has grown as additional cases have been reported. As of 24 January 2020, with reports of thirteen exportation events, the cumulative incidence in China is estimated at 5502 cases (95% confidence interval: 3027, 9057).
Our latest estimate is comparable to a preliminary report posted by a research group at Imperial College London (ICL) on their own homepage on 22 January 2020 [26] that estimated the incidence based on three importation events at 4000 cases (95% CI: 1000, 9700). Possible reasons for the slight difference include (i) the number of travelers in the previous study was derived from airline passenger data [27] and (ii) the assumed length of T was different. Two other estimates have also been published: a preliminary study by a Northeastern University group estimated 1250 cases (95% CI: 350, 3000) as of 17 January 2020 [28] and a University of Hong Kong group estimated 1343 cases (95% CI: 547, 3446) as of 17 January 2020 [29] . The former study from the United States assumes that the catchment area population is 10 million (we use 11.1 million).
The number of reported 2019-nCoV infections continues to grow as surveillance and detection methods improve. Our estimate and others [26, 28, 29] agree that the actual number of cases is likely in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than that of the originally linked seafood market in Wuhan have occurred. Such exposures are expected to include human-to-human transmission, but the levels of transmissibility have yet to be quantified. It is still plausible that a substantial number of human infections arose from animal-to-human exposures, such as was the case during the first outbreak of highly pathogenic influenza (H7N9) in China, 2013, and the human-to-human transmissibility has yet to be quantified in an explicit manner.
Despite initially restricting what information on the outbreak was shared publicly, the Chinese government has begun to respectfully provide updates on the situation on a daily basis. This encourages the real-time release of information by means of regularly updated situation reports, including epidemiological information with dates of exposure, illness onset, and hospitalization among cases.
For researchers to be able to contribute to control efforts by improving situation awareness via an explicit risk assessment, it is crucial that detailed epidemiological data are posted to a public domain in real-time. Such datasets should include not only a deidentified line list of cases but also updates on the infection status of traced contacts. Information on exposure period and illness onset can assist with the estimation of important natural history parameters such as the incubation period. It is critical for the public health community and the public at large to understand more about the process of case ascertainment, including the current case definition and reporting system mechanisms.
The authors declare no conflicts of interest. | 2,554 | When was the a cluster of pneumonia cases were first reported ? | {
"answer_start": [
1124
],
"text": [
"31 December 2019,"
]
} | 1,235 |
817 | The Extent of Transmission of Novel Coronavirus in Wuhan, China, 2020
https://doi.org/10.3390/jcm9020330
SHA: 919c524f19f79213e6f81aa38502c70287d273dc
Authors: Nishiura, Hiroshi; Jung, Sung-mok; Linton, Natalie M.; Kinoshita, Ryo; Yang, Yichi; Hayashi, Katsuma; Kobayashi, Tetsuro; Yuan, Baoyin; Akhmetzhanov, Andrei R.
Date: 2020
DOI: 10.3390/jcm9020330
License: cc-by
Abstract: A cluster of pneumonia cases linked to a novel coronavirus (2019-nCoV) was reported by China in late December 2019. Reported case incidence has now reached the hundreds, but this is likely an underestimate. As of 24 January 2020, with reports of thirteen exportation events, we estimate the cumulative incidence in China at 5502 cases (95% confidence interval: 3027, 9057). The most plausible number of infections is in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than the one in the epidemiologically linked seafood market in Wuhan have occurred.
Text: Since the announcement of a cluster of pneumonia cases of unknown etiology in Wuhan, Hubei Province, China, was made on 31 December 2019, many rapid virological, clinical, and epidemiological research responses have taken place [1, 2] . The causative agent of the pneumonia is suggested to be a novel coronavirus (2019-nCoV) of the same lineage (but genetically distinct) from the coronavirus causing severe acute respiratory syndrome (SARS) [1] . Cases in the initial cluster reported a common exposure-a seafood market in Wuhan where wild animals were served at a restaurant-indicating that a point-source zoonotic (animal-to-human) route was likely the main mode of transmission for those cases [2] .
Although early reports from Wuhan [3] stated that (i) there were only tens of cases in the cluster and (ii) no human-to-human transmission was directly observed, the scientific community was alert to the possibility that the novel coronavirus would spread to other geographic locations-including other countries-via direct human-to-human transmission. In early January, the outbreak began to escalate rapidly with hundreds of cases now confirmed along with the presence of a few household clusters [4] [5] [6] [7] .
As of 24 January 2020, the cumulative incidence in China is 830 cases, of which 549 cases were diagnosed in Hubei, 26 in Beijing, 20 in Shanghai, and 53 in Guangdong. Additionally, twenty-six deaths have been linked to the outbreak [6, 8] , and thirteen cases were exported to Japan, Singapore, South Korea, Taiwan, Thailand, Vietnam and the United States as of 22 January 2020. Considering that enhanced surveillance has been underway in these importing countries, case ascertainment has been perhaps better in exported case data.
Using a spatial back-calculation method and analyzing exported cases, we estimate the cumulative incidence of 2019-nCoV cases in China in real time, allowing us to update and discuss the extent of transmission at the source. Table 1 shows the incidence of exported cases by date of hospitalization and report. Due to the initial difficulty of diagnosis in the absence of established primer for polymerase chain reaction testing, the time lag between hospitalization and reporting was longer for early cases compared with that of more recent cases. Among the seven locations reporting importation, the total volume of inbound passengers from China was m = 63.1 million per year in 2017 [9] , of which 100q = 2.1% were from Wuhan [10] , a home of n = 19.0 million people as the catchment population of Wuhan airport. Two other locations with confirmed cases, i.e., Macau and Hong Kong, were excluded from the analysis, because it is commutable by land transporation and the first case in Hong Kong was indeed not via airtravel. As we already know from elsewhere [11] [12] [13] , given the observed cumulative count of c exported cases, we have a balance equation of the cumulative risk of infection:
where T is the sum of incubation and infectious periods, and here is assumed to be 3.2 and 9.3 days [14] , respectively, assuming that these periods are similar to those of other coronaviruses, and thus, T = 12.5 days. The estimated incidence in China is then given bypn. With an ad-hoc assumption that the data are generated following the binomial sampling process among travelers from Wuhan, the cumulative incidence is then estimated using a maximum likelihood method. Table 1 also shows the estimated incidence in China. The first exportation event in Thailand suggests 423 cases with the upper confidence limit of 1863 cases. The estimated cumulative incidence has grown as additional cases have been reported. As of 24 January 2020, with reports of thirteen exportation events, the cumulative incidence in China is estimated at 5502 cases (95% confidence interval: 3027, 9057).
Our latest estimate is comparable to a preliminary report posted by a research group at Imperial College London (ICL) on their own homepage on 22 January 2020 [26] that estimated the incidence based on three importation events at 4000 cases (95% CI: 1000, 9700). Possible reasons for the slight difference include (i) the number of travelers in the previous study was derived from airline passenger data [27] and (ii) the assumed length of T was different. Two other estimates have also been published: a preliminary study by a Northeastern University group estimated 1250 cases (95% CI: 350, 3000) as of 17 January 2020 [28] and a University of Hong Kong group estimated 1343 cases (95% CI: 547, 3446) as of 17 January 2020 [29] . The former study from the United States assumes that the catchment area population is 10 million (we use 11.1 million).
The number of reported 2019-nCoV infections continues to grow as surveillance and detection methods improve. Our estimate and others [26, 28, 29] agree that the actual number of cases is likely in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than that of the originally linked seafood market in Wuhan have occurred. Such exposures are expected to include human-to-human transmission, but the levels of transmissibility have yet to be quantified. It is still plausible that a substantial number of human infections arose from animal-to-human exposures, such as was the case during the first outbreak of highly pathogenic influenza (H7N9) in China, 2013, and the human-to-human transmissibility has yet to be quantified in an explicit manner.
Despite initially restricting what information on the outbreak was shared publicly, the Chinese government has begun to respectfully provide updates on the situation on a daily basis. This encourages the real-time release of information by means of regularly updated situation reports, including epidemiological information with dates of exposure, illness onset, and hospitalization among cases.
For researchers to be able to contribute to control efforts by improving situation awareness via an explicit risk assessment, it is crucial that detailed epidemiological data are posted to a public domain in real-time. Such datasets should include not only a deidentified line list of cases but also updates on the infection status of traced contacts. Information on exposure period and illness onset can assist with the estimation of important natural history parameters such as the incubation period. It is critical for the public health community and the public at large to understand more about the process of case ascertainment, including the current case definition and reporting system mechanisms.
The authors declare no conflicts of interest. | 2,554 | What is the number of inbound passengers from China? | {
"answer_start": [
3413
],
"text": [
" 63.1 million per year in 2017 "
]
} | 1,236 |
818 | The Extent of Transmission of Novel Coronavirus in Wuhan, China, 2020
https://doi.org/10.3390/jcm9020330
SHA: 919c524f19f79213e6f81aa38502c70287d273dc
Authors: Nishiura, Hiroshi; Jung, Sung-mok; Linton, Natalie M.; Kinoshita, Ryo; Yang, Yichi; Hayashi, Katsuma; Kobayashi, Tetsuro; Yuan, Baoyin; Akhmetzhanov, Andrei R.
Date: 2020
DOI: 10.3390/jcm9020330
License: cc-by
Abstract: A cluster of pneumonia cases linked to a novel coronavirus (2019-nCoV) was reported by China in late December 2019. Reported case incidence has now reached the hundreds, but this is likely an underestimate. As of 24 January 2020, with reports of thirteen exportation events, we estimate the cumulative incidence in China at 5502 cases (95% confidence interval: 3027, 9057). The most plausible number of infections is in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than the one in the epidemiologically linked seafood market in Wuhan have occurred.
Text: Since the announcement of a cluster of pneumonia cases of unknown etiology in Wuhan, Hubei Province, China, was made on 31 December 2019, many rapid virological, clinical, and epidemiological research responses have taken place [1, 2] . The causative agent of the pneumonia is suggested to be a novel coronavirus (2019-nCoV) of the same lineage (but genetically distinct) from the coronavirus causing severe acute respiratory syndrome (SARS) [1] . Cases in the initial cluster reported a common exposure-a seafood market in Wuhan where wild animals were served at a restaurant-indicating that a point-source zoonotic (animal-to-human) route was likely the main mode of transmission for those cases [2] .
Although early reports from Wuhan [3] stated that (i) there were only tens of cases in the cluster and (ii) no human-to-human transmission was directly observed, the scientific community was alert to the possibility that the novel coronavirus would spread to other geographic locations-including other countries-via direct human-to-human transmission. In early January, the outbreak began to escalate rapidly with hundreds of cases now confirmed along with the presence of a few household clusters [4] [5] [6] [7] .
As of 24 January 2020, the cumulative incidence in China is 830 cases, of which 549 cases were diagnosed in Hubei, 26 in Beijing, 20 in Shanghai, and 53 in Guangdong. Additionally, twenty-six deaths have been linked to the outbreak [6, 8] , and thirteen cases were exported to Japan, Singapore, South Korea, Taiwan, Thailand, Vietnam and the United States as of 22 January 2020. Considering that enhanced surveillance has been underway in these importing countries, case ascertainment has been perhaps better in exported case data.
Using a spatial back-calculation method and analyzing exported cases, we estimate the cumulative incidence of 2019-nCoV cases in China in real time, allowing us to update and discuss the extent of transmission at the source. Table 1 shows the incidence of exported cases by date of hospitalization and report. Due to the initial difficulty of diagnosis in the absence of established primer for polymerase chain reaction testing, the time lag between hospitalization and reporting was longer for early cases compared with that of more recent cases. Among the seven locations reporting importation, the total volume of inbound passengers from China was m = 63.1 million per year in 2017 [9] , of which 100q = 2.1% were from Wuhan [10] , a home of n = 19.0 million people as the catchment population of Wuhan airport. Two other locations with confirmed cases, i.e., Macau and Hong Kong, were excluded from the analysis, because it is commutable by land transporation and the first case in Hong Kong was indeed not via airtravel. As we already know from elsewhere [11] [12] [13] , given the observed cumulative count of c exported cases, we have a balance equation of the cumulative risk of infection:
where T is the sum of incubation and infectious periods, and here is assumed to be 3.2 and 9.3 days [14] , respectively, assuming that these periods are similar to those of other coronaviruses, and thus, T = 12.5 days. The estimated incidence in China is then given bypn. With an ad-hoc assumption that the data are generated following the binomial sampling process among travelers from Wuhan, the cumulative incidence is then estimated using a maximum likelihood method. Table 1 also shows the estimated incidence in China. The first exportation event in Thailand suggests 423 cases with the upper confidence limit of 1863 cases. The estimated cumulative incidence has grown as additional cases have been reported. As of 24 January 2020, with reports of thirteen exportation events, the cumulative incidence in China is estimated at 5502 cases (95% confidence interval: 3027, 9057).
Our latest estimate is comparable to a preliminary report posted by a research group at Imperial College London (ICL) on their own homepage on 22 January 2020 [26] that estimated the incidence based on three importation events at 4000 cases (95% CI: 1000, 9700). Possible reasons for the slight difference include (i) the number of travelers in the previous study was derived from airline passenger data [27] and (ii) the assumed length of T was different. Two other estimates have also been published: a preliminary study by a Northeastern University group estimated 1250 cases (95% CI: 350, 3000) as of 17 January 2020 [28] and a University of Hong Kong group estimated 1343 cases (95% CI: 547, 3446) as of 17 January 2020 [29] . The former study from the United States assumes that the catchment area population is 10 million (we use 11.1 million).
The number of reported 2019-nCoV infections continues to grow as surveillance and detection methods improve. Our estimate and others [26, 28, 29] agree that the actual number of cases is likely in the order of thousands, rather than hundreds, and there is a strong indication that untraced exposures other than that of the originally linked seafood market in Wuhan have occurred. Such exposures are expected to include human-to-human transmission, but the levels of transmissibility have yet to be quantified. It is still plausible that a substantial number of human infections arose from animal-to-human exposures, such as was the case during the first outbreak of highly pathogenic influenza (H7N9) in China, 2013, and the human-to-human transmissibility has yet to be quantified in an explicit manner.
Despite initially restricting what information on the outbreak was shared publicly, the Chinese government has begun to respectfully provide updates on the situation on a daily basis. This encourages the real-time release of information by means of regularly updated situation reports, including epidemiological information with dates of exposure, illness onset, and hospitalization among cases.
For researchers to be able to contribute to control efforts by improving situation awareness via an explicit risk assessment, it is crucial that detailed epidemiological data are posted to a public domain in real-time. Such datasets should include not only a deidentified line list of cases but also updates on the infection status of traced contacts. Information on exposure period and illness onset can assist with the estimation of important natural history parameters such as the incubation period. It is critical for the public health community and the public at large to understand more about the process of case ascertainment, including the current case definition and reporting system mechanisms.
The authors declare no conflicts of interest. | 2,554 | What percent of inbound passengers from China were from Wuhan? | {
"answer_start": [
3466
],
"text": [
"2.1%"
]
} | 1,237 |
819 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What cellular process is the Tat protein essential to? | {
"answer_start": [
479
],
"text": [
"HIV-1 replication"
]
} | 5,128 |
820 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Where does the Tat protein move to in cells? | {
"answer_start": [
519
],
"text": [
"nucleoplasm and the nucleolus"
]
} | 5,129 |
821 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What is a nucleolus? | {
"answer_start": [
565
],
"text": [
"a highly dynamic and structured membrane-less sub-nuclear compartment"
]
} | 5,130 |
822 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Where are rRNA and ribosomes created? | {
"answer_start": [
554
],
"text": [
"nucleolus"
]
} | 5,131 |
823 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | How many proteins were shown to change the amount of Jurkat T-cell nucleolus significantly? | {
"answer_start": [
1433
],
"text": [
"49"
]
} | 5,132 |
824 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What cellular processes occur in the nucleolus? | {
"answer_start": [
2985
],
"text": [
"RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly"
]
} | 5,133 |
825 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Which viruses target the nucleolus as part of their replication strategy? | {
"answer_start": [
4678
],
"text": [
"HIV-1, hCMV, HSV and KSHV"
]
} | 5,134 |
826 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What nucleolar antigen is essential of localization of Tat and Rev proteins? | {
"answer_start": [
5436
],
"text": [
"B23"
]
} | 5,135 |
827 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What was studied in this report? | {
"answer_start": [
6845
],
"text": [
"systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells"
]
} | 5,136 |
828 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What was studied in this report? | {
"answer_start": [
7434
],
"text": [
"the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart,"
]
} | 5,137 |
829 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Which isotope labeled arginine? | {
"answer_start": [
9657
],
"text": [
"light (R0K0)"
]
} | 5,138 |
830 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Which isotope labeled lysine? | {
"answer_start": [
9674
],
"text": [
"heavy (R6K6)"
]
} | 5,139 |
831 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | How many cells were harvested from each culture? | {
"answer_start": [
9799
],
"text": [
"85 million"
]
} | 5,140 |
832 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | How long is the nuclear protein PARP-1? | {
"answer_start": [
10640
],
"text": [
"113 kDa"
]
} | 5,141 |
833 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | How long is the protein Alpha-tubulin? | {
"answer_start": [
10762
],
"text": [
"50 kDa"
]
} | 5,142 |
834 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Where was alpha-tubulin found most abundantly in the cell? | {
"answer_start": [
10797
],
"text": [
"cytoplasmic"
]
} | 5,143 |
835 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | Where was alpha-tubulin found least abundantly in the cell? | {
"answer_start": [
10845
],
"text": [
"nuclear"
]
} | 5,144 |
836 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What is shown in Table S1? | {
"answer_start": [
11360
],
"text": [
"The fully annotated list of the quantified nucleolar proteins"
]
} | 5,145 |
837 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What types of cells are used to study Tat-mediated pathogenesis? | {
"answer_start": [
32303
],
"text": [
"Jurkat T-cells"
]
} | 5,146 |
838 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | How many proteins displayed a significant fold change? | {
"answer_start": [
34212
],
"text": [
"49"
]
} | 5,147 |
839 | Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/
SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9
Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.
Date: 2012-11-15
DOI: 10.1371/journal.pone.0048702
License: cc-by
Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] .
The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] .
The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions.
Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated.
In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis.
In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ).
We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination.
Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus.
We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold.
Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes.
We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression.
Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] .
Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] .
STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] .
YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] .
ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] .
Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] .
CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] .
ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] .
To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold).
In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) .
Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF.
Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] .
Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression.
Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) .
DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) .
Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] .
Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro.
The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function.
We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag.
Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] .
Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis.
HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability.
Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection.
Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] .
HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules.
We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined.
The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach.
The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours.
While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention.
Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore).
The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter).
Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology).
For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] .
Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap.
The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown).
The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%.
Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] .
The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific).
To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective.
The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key:
(R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis.
(DOCX) | 1,686 | What is the significance of this study? | {
"answer_start": [
45092
],
"text": [
"the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression"
]
} | 5,148 |
840 | The influenza pandemic preparedness planning tool InfluSim
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1832202/
SHA: f3f471d10a36a7a28e9050c10bd4dfd680cba17b
Authors: Eichner, Martin; Schwehm, Markus; Duerr, Hans-Peter; Brockmann, Stefan O
Date: 2007-03-13
DOI: 10.1186/1471-2334-7-17
License: cc-by
Abstract: BACKGROUND: Planning public health responses against pandemic influenza relies on predictive models by which the impact of different intervention strategies can be evaluated. Research has to date rather focused on producing predictions for certain localities or under specific conditions, than on designing a publicly available planning tool which can be applied by public health administrations. Here, we provide such a tool which is reproducible by an explicitly formulated structure and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. RESULTS: InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations, deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java, operates platform independent and can be executed on regular desktop computers. CONCLUSION: InfluSim is an online available software which efficiently assists public health planners in designing optimal interventions against pandemic influenza. It can reproduce the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability.
Text: Preparedness against pandemic influenza has become a high priority public health issue and many countries that have pandemic preparedness plans [1] . For the design of such plans, mathematical models and computer simulations play an essential role because they allow to predict and compare the effects of different intervention strategies [2] . The outstanding significance of the tools for purposes of intervention optimization is limited by the fact that they cannot maximize realism, generality and precision at the same time [3] . Public health planners, on the other hand, wish to have an optimal combination of these properties, because they need to formulate intervention strategies which can be generalized into recommendations, but are sufficiently realistic and precise to satisfy public health requirements.
Published influenza models which came into application, are represented by two extremes: generalized but oversimplified models without dynamic structure which are publicly available (e.g. [4] ), and complex computer simulations which are specifically adjusted to real conditions and/or are not publicly available (e.g. [5, 6] ). The complexity of the latter simulations, however, is not necessary for a reliable description of infection dynamics in large populations [7] . A minimum requirement for a pandemic influenza planning tool is a dynamic modelling structure which allows investigation of time-dependent variables like incidence, height of the epidemic peak, antiviral availability etc. The tool should, on the other hand, be adjustable to local conditions to adequately support the pandemic preparedness plans of different countries which involve considerably different assumptions (Table 1) .
Here we describe a publicly available influenza pandemic preparedness planning tool [8] which is designed to meet the requirements in preparedness planning. It is based on an explicitly formulated dynamic system which allows addressing time-dependent factors. It is sufficiently flexible to evaluate the impact of most candidate interventions and to consider local conditions like demographic and economic factors, contact patterns or constraints within the public health system. In subsequent papers we will also provide examples and applications of this model for various interventions, like antiviral treatment and social distancing measures.
The model is based on a system of 1,081 differential equations which extend the classic SEIR model. Demographic parameters reflect the situation in Germany in 2005, but can be adjusted to other countries. Epidemiologic and clinic values were taken from the literature (see Tables 1, 2 , 3, 4, 5, 6 and the sources quoted there). Pre-set values can be varied by sliders and input fields to make different assumptions on the transmissibility and clinical severity of a new pandemic strain, to change the costs connected to medical treatment or work loss, or to simply apply the simulation to different demographic settings. Model properties can be summarized as follows. The mathematical formulation of this model is presented in detail in the online supporting material. The corresponding source code, programmed in Java, and further information can be downloaded from [8] .
According to the German National Pandemic Preparedness Plan [9] , the total population is divided in age classes, each of which is subdivided into individuals of low and high risk ( Table 2) . Transmission between these age classes is based on a contact matrix (Table 3) which is scaled such that the model with standard parameter values yields a given basic reproduction number R0. Values for the R0 associated with an influenza strain with pandemic potential are suggested to lie between 2 and 3 [10] . This value is higher than the effective reproduction number which has been estimated to be slightly lower than 2 [11, 12] . As a standard parameter, we use R0 = 2.5 which means that cases infect on average 2.5 individuals if everybody is susceptible and if no interventions are performed.
Susceptible individuals who become infected, incubate the infection, then become fully contagious and finally develop protective immunity (Table 4) . A fraction of cases remains asymptomatic; others become moderately sick or clinically ill (i.e. they need medical help). Depending on the combination of age and risk group, a fraction of the clinically ill cases needs to be hospitalized, and an agedependent fraction of hospitalized cases may die from the disease ( Table 5 ). This partitioning of the cases into four categories allows combining the realistic description of the transmission dynamics with an easy calculation of the resources consumed during an outbreak. The degree and duration of contagiousness of a patient depend on the course of the disease; the latter furthermore depends on the age of the patient (Table 5) . Passing through the incubation and contagious period is modelled in several stages which allows for realistic distributions of the sojourn times ( Table 4 ). The last two stages of the incubation period are used as early infectious period during which the patient can already spread the disease. Infectiousness is highest after onset of symptoms and thereafter declines geometrically (Table 6 ). Clinically ill patients seek medical help on average one day after onset of symptoms. Very sick patients are advised to withdraw to their home until their disease is over, whereas extremely sick patients need to be hospitalized and may die from the disease (Table 4) . After the end of their contagious period, clinically ill patients go through a convalescent period before they can resume their ordinary life and go back to work (Table 4) .
We provide some examples of model output of InfluSim [8] , version 2.0, by means of four sensitivity analyses; further investigations will be presented elsewhere. Figure 1 shows the graphical user interface of the software which is divided into input and output windows. The user may set new values in the input fields or move sliders to almost simultaneously obtain new results for the course of an epidemic in a given population. Figures 2A and 2B show pandemic waves which result from varying the basic reproduction number from 1.5 to 4.0. Using the standard parameter values as given in Tables 2, 3 , 4, 5, 6 and omitting all interventions in a town of 100,000 inhabitants results in a pandemic wave which lasts for about ten weeks (Figure 2A , with R 0 = 2.5). The peak of the pandemic wave is reached after six to seven weeks, with a daily incidence of up to 2,340 influenza patients seeking medical help, with up to 280 hospital beds occupied by influenza cases and with up to 14,000 out of 60,000 working adults unable to go to work because of illness or convalescence. These results depend on the assumptions concerning the yet unknown contagiousness and pathogenicity of the virus. Figures 2C and 2D show how the shape of the curves depends on the course of contagiousness: the pandemic wave proceeds relative slowly if the contagiousness does not change during the infectious period (x 50 = 50%), but proceeds quickly if the contagiousness is highest after onset of symptoms and decreases thereafter (x 50 > 50%).
The influenza pandemic preparedness planning tool InfluSim stands between simple spreadsheet models and sophisticated stochastic computer simulations. It describes a pandemic wave within a homogeneously mixing population like a town or city, but surprisingly produces the same dynamics as individual-based simulations which explicitly consider geographic spread through the US (cf. [6] and [5] with Figure 2 using R 0 = 2). Similar observations were made with a simple deterministic compartmental model [7] . Stochastic models are known to behave quasi-deterministically when the simulated population becomes very large.
A further reason for the congruence of complex stochastic and simple deterministic models must lie in the incredi-bly quick way in which pandemic influenza spreads geographically. Unless being controlled at the place of origin [12, 13] , a pandemic starting in a far-off country will lead to multiple introductions [14] into the large industrialized nations where it can be expected to quickly spread to neighbouring towns and to rural areas. The large populations which have to be considered susceptible to a pandemic virus and the quick geographic spread tend to diminish the differences between the results of sophisticated individual-based and simple deterministic models.
However, a deterministic model like InfluSim cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities. Examples are: (i) a geographically limited spread and fairly effective control measures can imply that the epidemic affects only a small population and thus, may be strongly influenced by stochastic events [15] [16] [17] ; (ii) transmission which predominantly occurs in households or hospitals, or which is driven by other substantial features of the contact network is not in agreement with the assumption of homogeneous mixing in the deterministic model cannot reliably predict the spread of infection [18] [19] [20] [21] [22] [23] . In particular, (iii) super-spreading events can substantially change the course of an epidemic compared to the deterministic prediction [24] [25] [26] [27] . Apart from such factors, the predictability of intervention success is generally subject to uncertainties in the choice of parameter values, Assumed scenarios and outcomes of pandemic preparedness plans. * Gross attack rate (i.e. clinically ill and moderately ill cases). A population of N = 100,000 inhabitants of Germany is subdivided according to age a and risk category r. We assume that all age groups are fully susceptible at begin of the outbreak. A fraction of F a = 6% of all children (age < 20 years) are regarded as being under high risk (r = r 1 ) after an influenza infection whereby the remaining 94% are under low risk (r = r 2 ). The high risk fractions of working adults (ages 20-59) and elderly (ages 60+) are F a = 14% and F a = 47%, respectively. Source: [9] demanding additional efforts like Bayesian approaches [28] to evaluate the reliability of predictions [29] .
Pandemic preparedness plans must consider constraints and capacities of locally operating public health systems. The time-dependent solutions of InfluSim allow assessing peak values of the relevant variables, such as outpatients, hospitalizations and deaths. Various interventions may be combined to find optimal ways to reduce the total number of cases, to lower the peak values or to delay the peak, hoping that at least part of the population may benefit from a newly developed vaccine.
Special care was taken when implementing a variety of pharmaceutical and non-pharmaceutical interventions which will be discussed in subsequent papers. Despite its comprehensible structure, the model does not suffer from over-simplifications common to usual compartment models. Instead of implicitly using exponentially distributed sojourn times, we have implemented realistically distributed delays. For example, the model considers that individuals may transmit infection before onset of symptoms, and that some cases may remain asymptomatic, but still infecting others. Such features have serious implications for the success of targeted control measures.
InfluSim is freely accessible, runs on a regular desktop computer and produces results within a second after changing parameter values. The user-friendly interface and the ease at which results can be generated make this program a useful public health planning tool. Although we have taken care of providing a bug-free program, including the source code, the user is encouraged to treat results with due caution, to test it, and to participate in bug-reports and discussions on the open-source platform [30] which also provides regular updates of InfluSim.
The author(s) declare that they have no competing interests.
ME developed the model, MS designed the software, HPD wrote the manuscript and SOB formulated the public The who-acquires-infection-from-whom matrix shows the frequency of contacts (per week per person) between different age classes.
Source: [38] . Distribution of sojourn times (the last two stages of the latent period are used as early infectious period with an average duration of D L = 0.5 days). Sources: A [11] , B [39, 40] , C assumed, D [41] health requirements of the software. All authors read and approved the final manuscript.
Susceptible individuals S a, r are infected at a rate λ a (t) which depends on their age a and on time t. Infected individuals, E a, r , incubate the infection for a mean duration D E . To obtain a realistic distribution of this duration, the incubation period is modelled in n stages so that progression from one stage to the next one occurs at rate δ = n/D E .
The last l incubation stages are regarded as early infectious period during which patients may already spread the infection (this accounts for an average time of lD E /n for the "early infectious period" which is about half a day for the standard set of parameters). After passing through the last incubation stage, infected individuals become fully contagious and a fraction of them develops clinical symptoms. The course of disease depends on the age a of the infected individual and on the risk category r to which he or she belongs: a fraction c a, r (A) becomes asymptomatic (A a ), a fraction c a, r (M) becomes moderately sick (M a ), a fraction c a, r (V) becomes very sick (V a ) and the remaining fraction c a, r (X) becomes extremely sick (X a ) and need hospitalization (i.e., c a, r (A) + c a, r (M) + c a, r (V) + c a, r (X) = 1 for each combination of a and r). ) . A fraction f V (t) of all severe and a fraction f X (t) of all extremely severe cases who visit the doctor within D T days after onset of symptoms are offered antiviral treatment, given that its supply has not yet been exhausted. As our model does not explicitly consider the age of the disease (which would demand partial differential equations), we use the contagious stages to measure time since onset and allow for treatment up to stage m a, T Sources: Contagiousness of asymptomatic cases: [11] ; degree of contagiousness during the early infectious period and equality of the contagiousness of moderately and severely sick cases: assumed. Independent of age a and risk group r, a fraction c a, r (A) = 33% of infections result in asymptomatic cases, a fraction c a, r (M) = 33.5% become moderately sick and the remaining fraction develops severe disease. An age-and risk-dependent fraction h a, r of untreated patients with severe disease needs hospitalization. An age-dependent fraction d a of hospitalized cases dies. Sources: fraction of asymptomatic cases: [11] ; 50% of symptomatic cases see a doctor: [9] ; hospitalizations per severe case: [9] ; case fatality of hospitalized, but untreated patients calculated from [4] .
(see below for details). This imposes some variability to the maximum time until which treatment can be given, which may even improve the realism of the model with respect to real-life scenarios. Antiviral treatment reduces the patients' contagiousness by f I percent and it reduces hospitalization and death by f H percent. Extremely sick patients, whose hospitalization is prevented by treatment, are sent home and join the group of treated very sick patients(W a, T ). The remaining duration of disease and contagiousness of treated cases is reduced by f D percent so that their rate of progressing from one stage to the next has To obtain a realistic distribution of this sojourn time, convalescence is modelled in j stages so that progression from one stage to the next occurs at rate ρ = j/D C . Fully recovered patients who have passed through their last stage of convalescence join the group of healthy immunes I; working adults will go back to work. Further interventions, describing the reduction of contacts, will be discussed after the presentation of the differential equations.
InfluSim user interface Figure 1 InfluSim user interface. x 50 = 95% means that 95% of the cumulative contagiousness is concentrated during the first half of the contagious period, see Table 6 ). D: Cumulative number of deaths for values of x 50 as in C. All other parameters as listed in Tables 2-6 . Hospitalized, but untreated cases
Contact matrix For the mixing of the age classes, we employ a whoacquires-infection-from whom matrix which gives the relative frequency of contacts of infective individuals of age a i with other people of age a s . In this paper, we assume bi-directional contacts (e.g. children have the same total number of contacts with adults as adults with children). Multiplication of this matrix with an appropriate constant scaling factor κ (see below)
results in the matrix of crude contact rates .
In the absence of interventions, we have to multiply these contact rates with the contagiousness factors b L , b A , b M and b V to obtain the effective contact rates:
during the early infectious period, of asymptomatic cases, of moderately sick cases, of (untreated) very sick cases.
To assess the effect of day care centre and school closing on the transmission of an infectious disease, we have to first make an assumption on what fraction r sch of the contacts among healthy children who are in the same age class occurs in day care centres and schools. The contact rates between very sick or hospitalized children (who do not attend day care centre or school) and other children need, therefore, be reduced to (contact rate between healthy and very sick children in the same age class, i.e. a i = a s ).
As very sick children have to be taken care of by adults at home or in hospital, their contact rate to adults increases by a factor F HC (contact rate between very sick children of age a i and adults of age a s ).
Contacts between very sick children and other children in a higher or lower age class remain unchanged: (contact rate between healthy children of age a s and very sick children of a different age a i ).
Closing day care centres and schools at time t will not necessarily prevent all the contacts that would have happened with other children. During the closing of schools and day care centres, the contact rates between susceptible children of age a s and infected children of age a i who are in their late incubation period ( ), who are asymptomatic ( ), or who are moderately sick ( ) are reduced by the factor r sch if the children are in the same age class:
where 1 sch (t) is a function which indicates when schools and day care centres are opened or closed:
,..., While day care centres and schools are closed, children (age a i ) need adult supervision at home. Their contact with susceptible adults (age a s ) increases by the "child care factor" F CC :
Child care at home also increases the exposure of healthy children (age a s ) to contagious adults (age a i ):
Cancelling mass gathering events effects only the contacts of adults who are healthy enough to attend such events. Assuming that such an intervention at time t reduces contacts by a fraction r mass , we get for all contacts between susceptible adults of age a s and infectious adults of age a i the following contact rates:
where 1 mass (t) is a function which indicates when mass gathering events are possible or when they are closed:
As contacts with adults who are too sick to attend such mass gathering events cannot be prevented by this measure it is .
During some time in the epidemic, the general population may effectively reduce contacts which can be a result of wearing facial masks, increasing "social distance", adopting improved measures of "respiratory hygiene" or simply of a general change in behaviour. This will be implemented in the program by reducing the contacts of susceptible individuals at that time t by factor r gen (t
while mass gathering events are forbidden while m mass gathering events are allowed. while the population reduces their contacts while the population behaves as usual.
The contact rates of cases in the late incubation period and that of asymptomatic cases remain unchanged:
for infected individuals in the late incubation period, for asymptomatic cases.
To allow for a contagiousness which changes over the course of disease, we multiply each contact rate with a weighting factor whereby k is the stage of contagiousness. This leads to the following contact rates:
for asymptomatic cases in For x = 1, contagiousness is equally high in all stages; for x = 0, only the first stage is contagious; for 0 <x < 1, the contagiousness decreases in a geometric procession. We make the simplifying assumption that contagiousness does not change during the late incubation period for cases in stage k = n -l,..,n of the incubation period.
At time t = 0 and in the absence of interventions, the next generation matrix has the following elements where is the fraction of untreated extremely severe cases who die from the disease (see below for details). The dominant eigenvalue of this matrix is called the basic reproduction number R 0 . If κ (which determines the value of the contact rates ) is given, the eigenvectors of this matrix can numerically be calculated. The user-specified value of R 0 is now used to determine numerically the scaling factor κ. Let be the eigenvector which has the largest eigenvalue R 0 .
) ) −
Using the user-specified numbers of people N a in the age classes and the fractions F a of people under high risk within each age class (Table 2) , we obtain the initial population sizes according to age and risk class: Using these initial values, the set of differential equations is solved numerically with a Runge-Kutta method with step-size control.
if and in treatment window otherwise 0 ⎩ ⎩ | 1,600 | What are the limitations of a deterministic model? | {
"answer_start": [
10476
],
"text": [
"cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities"
]
} | 5,255 |
841 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What kinds of viruses are Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV)? | {
"answer_start": [
1906
],
"text": [
"arboviruses"
]
} | 3,003 |
842 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What are the current clinically-available methods to detect encephalitis viral antigens? | {
"answer_start": [
2177
],
"text": [
"ELISA and IFA"
]
} | 3,004 |
843 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What methods exist for detecting multiple antigens simultaneously in a one-sample, laboratory test? | {
"answer_start": [
2474
],
"text": [
"two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology"
]
} | 3,005 |
844 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | How many antigens could be detected by Liew's multiplex ELISA test? | {
"answer_start": [
2923
],
"text": [
"9"
]
} | 3,006 |
845 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What kind of antibodies were used in the ELISA-array assay? | {
"answer_start": [
3480
],
"text": [
"monoclonal"
]
} | 3,007 |
846 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | How was the ELISA assay validated? | {
"answer_start": [
3618
],
"text": [
"using cultured viruses and inoculated chicken eggs with patient sera"
]
} | 3,008 |
847 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What capture antibodies were used in the study? | {
"answer_start": [
4365
],
"text": [
"4D5, 2B5, 1F1, 2B8, 4F9, and 4E11"
]
} | 3,009 |
848 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | What was the spotting concentration range for the capture antibodies? | {
"answer_start": [
9091
],
"text": [
"from 0.2 to 0.0125 mg/ml"
]
} | 3,010 |
849 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | How was the proper spotting concentration determined? | {
"answer_start": [
9317
],
"text": [
"combination of minimized cross reaction and higher signal intensity"
]
} | 3,011 |
850 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | How was cross reaction detection determined? | {
"answer_start": [
9606
],
"text": [
"by applying JEV, YF, and DV cultures"
]
} | 3,012 |
851 | Development of an ELISA-array for simultaneous detection of five encephalitis viruses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3305475/
SHA: ef2b8f83d5a3ab8ae35e4b51fea6d3ed9eb49122
Authors: Kang, Xiaoping; Li, Yuchang; Fan, Li; Lin, Fang; Wei, Jingjing; Zhu, Xiaolei; Hu, Yi; Li, Jing; Chang, Guohui; Zhu, Qingyu; Liu, Hong; Yang, Yinhui
Date: 2012-02-27
DOI: 10.1186/1743-422x-9-56
License: cc-by
Abstract: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.
Text: Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), eastern equine encephalitis virus (EEEV), sindbis virus(SV), and dengue virus(DV) are arboviruses and cause symptoms of encephalitis, with a wide range of severity and fatality rates [1] . Establishment of an accurate and easy method for detection of these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, ELISA and IFA are the methods which are clinically-available for the detection of encephalitis viral antigens, but they could only detect one pathogen in one assay [2, 3] .
There are a variety of different methods available for identifying multiple antigens in one sample simultaneously, such as two-dimensional gel electrophoresis , protein chip, mass spectrometry, and suspension array technology [4] [5] [6] . However, the application of these techniques on pathogen detection is still in an early phase, perhaps due to the complicated use and high cost.
Antibody arrays for simultaneous multiple antigen quantification are considered the most accurate methods [7] [8] [9] [10] . Liew [11] validated one multiplex ELISA for the detection of 9 antigens; Anderson [12] used microarray ELISA for multiplex detection of antibodies to tumor antigens in breast cancer, and demonstrated that ELISA-based array assays had the broadest dynamic range and lowest sample volume requirements compared with the other assays.
However, the application of ELISA-based arrays is currently limited to detection of cancer markers or interleukins; no detection of pathogens has been reported. In this study, we developed an ELISA-based array for the simultaneous detection of five encephalitis viruses. Seven specific monoclonal antibodies were prepared against five encephalitis viruses and used to establish an ELISA-array assay. The assay was validated using cultured viruses and inoculated chicken eggs with patient sera. The results demonstrated that this method combined the advantage of ELISA and protein array (multiplex and ease of use) and has potential for the identification of clinical encephalitis virus.
Monoclonal antibodies were prepared from hybridoma cell lines constructed by Prof. Zhu et al. Purification was conducted by immunoaffinity chromatography on protein G affinity sepharose [13] . Specific monoclonal antibodies (4D5 against JEV, 2B5 against TBEV, 1F1 against SV, 2B8 against serotype 2 DV, 4F9 against serotype 4 DV, 4E11 against EEEV, and 2A10 against Flavivirus) were selected for this study. All of the antibodies were raised according to standard procedures.
Using 4D5, 2B5, 1F1, 2B8, 4F9, and 4E11 as capture antibodies, detection antibodies (2A10, 1 F1, and 4E11) were coupled to biotin-NHS ester(Pierce, Germany) at 4°C for 3 h according to the manufacturer's instructions. Unincorporated biotin was removed by Desalt spin column (Pierce). Immunologic reactions were reported by Streptavidin-HRP (CWBIO, Beijing, China) and Super Signal ELISA Femto Maximum sensitive substrate. Purified goat-anti mouse antibody was used as a positive control.
JEV and DV were cultured in C6/36 cells; SV, TBEV, and EEEV were cultured in BHK-21 cells. The culture of TBEV and EEEV was conducted in biosafety level 3 facility, however, JEV, DV and SV were conducted in biosafety level 2 facility. Viral titers were determined by the 50% tissue culture infectious dose (TCID 50 ) method. All the cultures were inactivated by 0.025% β-propionolactone at 4°C overnight, then 37°C for 1 h to decompose β-propionolactone.
Antibodies were spotted using a BIODOT machine (BD6000;California, USA) on ELISA plates (30 nl/dot). The plates were blocked with 3% BSA-PBS in 37°C for 1 h, followed by washing 3 times with PBS containing 0.1% Tween-20 for 2 min each. Then, the plates were dried, sealed, and stored at 4°C before use [11] .
When spotting, different spotting buffers and concentrations of capture monoclonal antibodies were evaluated to optimize the ELISA-array assay. The optimization was evaluated by dot morphology and signal intensity. The tested spotting buffers included 1 × phosphate buffer saline (PBS), PBS +20% glycerol, and 1 × PBS + 20% glycerol+0.004% Triton-X100. A range of monoclonal antibody concentrations (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml) were compared.
Following a double antibody sandwich format, printed plates were incubated sequentially with inactivated viral cultures, biotin-labeled detecting antibody, HPR-labeled avidin, and substrate, followed by signal evaluation.
Antigen binding was performed in PBS(containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h, followed by washing 3 times(1 × PBS containing 0.1% Tween-20). Incubation of ELISA plates with biotinylated detecting antibody cocktails was performed in PBS (containing 0.1% Tween-20 and 5% FCS) at 37°C for 2 h. After washing, specific binding of the detecting antibodies was reported by streptavidin-HRP and stained with Super Signal ELISA Femto Maximum sensitive substrate (Thermo scientific, Rockford, USA) [11, 14, 15] . Visualization of the plate was performed in AE 1000 cool CCD image analyzer(Beijing BGI GBI Biotech Company., LTD, China). The signal intensity and background of each spot was read out and recorded with "Monster"software. The positive signals were defined as a signal value > 400 and a signal value (sample)/signal value (negative) > 2.
The identical antibodies used in the ELISA-array format were also tested in a conventional ELISA format to determine the difference in sensitivity and specificity of the two methods. The conventional ELISAs were performed at the same time as the ELISA-array assays to ensure similar reaction conditions. The conventional ELISAs were performed in an identical maner to the ELISA-array, except that antibodies were coated at a concentration of 2 μg/mL in PBS (pH 7.4), and substrate TMB was used instead of Super Signal ELISA Femto Maximum sensitive substrate [16, 17] .
Three serum samples were collected from patients with nervous system symptoms and histories of tick bites. The serum samples were treated with penicillin and streptomycin, then inoculated into the allantoic cavities of chicken eggs. 3 days later, the liquid was collected and divided into two portions (one for inactivation and one for RNA extraction). The RNA and inactivated samples were stored at -70°C before use.
RNA was extracted from the inoculated chicken eggs using a RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. All RNA extraction procedures were conducted at BSL-3 facilities. The primers and probes were used as previously described [18] . The real-time RT-PCR was conducted with a Quti-teck q-RT-PCR Kit (Qiagen Inc,). The reaction consisted of 10 μL of 2 × reaction buffer (0.2 μL reverse transcription enzyme, and 250 nmol/l primers and probes). RNA and deionized water were added to a final volume of 20 μl. PCR was performed with a LightCycler 2.0 (Roche, Switzerland) [19] .
Optimization of the ELISA-array assay
The spotted array layout is depicted in Figure 1 and the efficacy of three different spotting buffers on the quality of the printed ELISA-arrays were investigated by spot morphology observation and signal intensity comparison.
The spotting concentration of the capture antibodies varied from 0.2 to 0.0125 mg/ml (each was serially diluted 2-fold). The efficacy of the spotting concentration of the capture antibodies was evaluated by virus culture detection, the proper spotting concentration was determined by a combination of minimized cross reaction and higher signal intensity. Figure 1 illustrates the array layout and Figure 2 demonstrates the result of the three spotting buffers and spot concentration of antibody 2B5 by TBE virus culture detection. Cross reaction detection was also conducted by applying JEV, YF, and DV cultures.
Spot morphology observation (Figures 2a, b , and 2c) demonstrated that spotting buffer containing PBS with 20% glycerol produced tailed spot morphology; buffers containing PBS alone and PBS with 20% glycerol +0.004% Triton-X100 gave good spot morphology (round and full). Buffers containing PBS with 20% glycerol and PBS with 20% glycerol+0.004% Triton-X100 produced higher signal intensities than PBS alone. Thus, PBS with 20% glycerol+0.004% Triton-X100 was adopted as the optimized spotting buffer for subsequent experiments. Simultaneously, the spot concentration evaluation suggested that 0.05 mg/ml was optimal. At this concentration, the signal intensity was higher and the cross-reaction did not appear (Figure 2d ). Consequently, spotting concentration optimization of other capture antibodies (4D5, 1F1, 4E11, and 2B8) demonstrated that 0.05 mg/ml was also suitable(data not shown).
The optimized ELISA array layout is shown in Figure 3 , which was applied in the following experiments.
Successful detection of viral pathogens requires a test with high sensitivity and specificity. To evaluate the performance of the designed antibody arrays, the specificity and sensitivity of the individual analytes were examined. By testing serially-diluted viral cultures, including DV-2, DV-4, JEV, TBE, SV, and EEEV, the sensitivity of ELISAarray and the identical conventional ELISA were compared ( Table 1 ). The detection limit of the two methods was compared and demonstrated. The cross-reactivity test was conducted using BHK-21 and vero cell lysate, Yellow fever virus (YFV) cultures (5 × 10 5 TCID 50 /ml, West Nile virus(WNV) cultures(2 × 10 6 TCID 50 /ml), and Western equine encephalitis virus(1 × 10 7 TCID 50 /ml). The results demonstrated that neither the ELISA-array nor traditional ELISA displayed cross-reactivity.
Equal volumes of cultured TBEV, JEV, DV-2, DV-4, SV, and EEEV were prepared for single sample detection; two or three of the cultures were mixed for multiplex detection. A cocktail of biotin conjugated antibody (2A10, 4E11, and 1F1) was used in all tests. The results demonstrated that for all virus combinations, each virus was detected specifically, with no false-positive or-negative results (Figures 4 and 5) .
Chicken eggs inoculated with infected human serum were used for validation of the ELISA-array assay. All samples showed high reaction signals with capture antibody 2B5, which was specific for TBEV ( Figure 6b ). The ELISA-array assay suggested that the three patients were all infected with TBEV.
To verify the results tested by ELISA-array, RNA extracted from chicken eggs was applied to a real time-RT-PCR assay using primers and probes targeting TBEV. The results were also positive (Figure 6a) . The consensus detection results confirmed that the ELISAarray assay was reliable.
To be widely used in the clinical setting, the detection system should be easy to use and can be performed by untrained staff with little laboratory and experimental experience. Moreover, when the volume of the clinical samples is limited and an increasing number of pathogens per sample needs to be tested, the detecting system should be high-throughput to allow detection of multiple pathogens simultaneously [6, 20, 21] . Multiple detection, easy to use, and affordability are requirements for detection methods in the clinical setting. Thus, an ELISA-array, which combines the advantages of ELISA and protein array, meets the above requirements.
It has been reported that an ELISA-array has been used in the diagnosis of cancer and auto-allergic disease [7, 12] ; however, No study has reported the detection of viral pathogens. In this study, we developed a multiplex ELISA-based method in a double-antibody sandwich format for the simultaneous detection of five encephalitis-associated viral pathogens.
The production of a reliable antibody chip for identification of microorganisms requires careful screening of capture of antibodies [14] . Cross-reactivity must be minimized and the affinity of the antibody is as important as the specificity. First, we prepared and screened 23 monoclonal antibodies against eight viruses and verified the specificity and affinity to the target viruses by an immunofluorescence assay. Then, the antibodies were screened by an ELISA-array with a double-antibody sandwich ELISA format. The antibodies which produced cross-reactivity and low-positive signals were excluded. Finally, six antibodies were selected as capture antibodies. Another monoclonal antibody, 2A10, which could specifically react with all viruses in the genus Flavivirus was used for detecting antibody against DV, JEV, and TBEV. For the detection of EEEV and SV, although the detecting and trapping antibodies were the same (1F1 and 4E11, respectively), the antibodies produced excellent positive signals. The epitope was not defined; however, we suspect that the antibodies both target the surface of the virions. As one virion exits as, many with the same epitope appear, thus no interference occurred using the same antibody in the double-antibody sandwich format assay.
Currently, the availability of antibodies suitable for an array format diagnostic assay is a major problem. In the ELISA-array assay, this problem exists as well. Because of the limitation of available antibodies, this assay could only detect 5 pathogens. In the future, with increasing numbers of suitable antibodies, especially specific antibodies against Flavivirus, this ELISAarray might be able to test more pathogens and be of greater potential use. To make the assay more amenable to multiple virus detection, the assay protocol was optimized. In addition to the dotting buffer, the capture antibody concentration and the different virus inactivation methods (heating and β-propiolactone) were also compared and evaluated. Heat inactivation was performed by heating the viral cultures at 56°C for 1 h, and β-propiolactone inactivation was performed by adding β-propiolactone into the retains better antigenicity than the heat-inactivation method. Thus, β-propiolactone treatment was chosen as the virus-inactivation method. A conventional ELISA is a standard method in many diagnostic laboratories. We compared the ELISA-array with a conventional ELISA and confirmed that the advantage of the ELISA-array was evident with comparable specificity and higher sensitivity than ELISA. The time required for the ELISA-array is significantly less than for conventional ELISA (4 h vs. a minimum of 6 h, respectively). Furthermore, less IgG is required for printing than for coating ELISA plates. Coating of a single well in microtiter plate requires 100 μl of a 1 μg/ml antibody solution, which is equivalent to 100 ng of IgG. For the ELISA-array, only 30 nl of a 50 μg/ml antibody solution is required for each spot, which is equivalent to 1.5 ng of IgG. With the characteristics of ease of use, sensitivity, specificity, and accuracy, the ELISA-array assay would be widely accepted for clinical use. | 1,553 | How was the ELISA-array assay validated? | {
"answer_start": [
3618
],
"text": [
"using cultured viruses and inoculated chicken eggs with patient sera"
]
} | 3,013 |
852 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | What is the role of antibodies during infection? | {
"answer_start": [
283
],
"text": [
"Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections."
]
} | 465 |
853 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | How can antibodies also create health problems? | {
"answer_start": [
488
],
"text": [
"Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. "
]
} | 467 |
854 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | Which technology invention produced antibodies that are clones of a unique parent cell? | {
"answer_start": [
2257
],
"text": [
" in the 1970s with the development of hybridoma technology to produce monoclonal antibodies "
]
} | 474 |
855 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | What mechanism is responsible for the creation of diversified repertoire for antibodies? | {
"answer_start": [
2372
],
"text": [
"somatic rearrangement during B cell differentiation was responsible for antibody diversification "
]
} | 476 |
856 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | What developments have been made possible by the study of B-cell repertoire? | {
"answer_start": [
2996
],
"text": [
"(1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease."
]
} | 478 |
857 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | What motivates the study of the rare B-cells that produce Broadly Neutralizing Antibodies (bnAb)? | {
"answer_start": [
3589
],
"text": [
"discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies"
]
} | 485 |
858 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | How has the study of B-cells helped the treatment for Respiratory syncytial virus (RSV)? | {
"answer_start": [
4083
],
"text": [
"For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates"
]
} | 489 |
859 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | How are the studies on B-cells helping the development of a universal influenza vaccine? | {
"answer_start": [
4315
],
"text": [
"Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine ("
]
} | 490 |
860 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | What role B-cell play in malaria infection and prevention? | {
"answer_start": [
4790
],
"text": [
" Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection"
]
} | 494 |
861 | Techniques to Study Antigen-Specific B Cell Responses
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/
SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c
Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .
With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61) . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79, 81, 82) . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding (26). Individual cells can also be isolated by fluorescence activated cell sorting (FACS) for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling.
Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry (20, 26, 80, 83) . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts (21, 26, 47, 50) . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry (Figure 1) . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: (1) Using decoys to exclude B cells of unwanted specificities;
(2) careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and (3) choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin.
In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs ( Table 2) , and antigen-specific B cells can be analyzed using the techniques described above.
Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits (2) . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy (62, 65) . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling (66) . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy (67, 68) . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells (isolated by limiting dilution or FACS) with two-photon microscopy (63, 69, 70) .
Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells (104) . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery (71) . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans (72) . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin (73) . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells (73, 74) . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection.
On the horizon, single cell RNA-sequencing (RNA-seq) technologies have the potential to revolutionize the study of antigen-specific immune cells (75, 76) . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics (105) (106) (107) .
The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role.
JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript. | 1,569 | How can the study of B-cells help in the prevention and treatment of autoimmune diseases? | {
"answer_start": [
6415
],
"text": [
"The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases."
]
} | 497 |
862 | Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/
SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b
Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.
Date: 2013-08-05
DOI: 10.1371/journal.pone.0069982
License: cc-by
Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).
Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms.
The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] .
Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] .
Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents.
The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform.
The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools.
N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php
The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads.
Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1-
The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation,
Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation,
The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme.
The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain.
For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.
After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times.
Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] .
Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain.
Type of inhibition Ki (mM) IC 50 (mM)
Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases.
These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d).
The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases.
The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors.
In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy.
Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness.
In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.
File S1 Types of inhibitions with Ki (Compounds 3a-3d).
(DOC) | 1,565 | What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms? | {
"answer_start": [
2276
],
"text": [
"cysteine proteases"
]
} | 3,041 |
863 | Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/
SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b
Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.
Date: 2013-08-05
DOI: 10.1371/journal.pone.0069982
License: cc-by
Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).
Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms.
The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] .
Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] .
Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents.
The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform.
The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools.
N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php
The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads.
Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1-
The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation,
Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation,
The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme.
The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain.
For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.
After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times.
Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] .
Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain.
Type of inhibition Ki (mM) IC 50 (mM)
Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases.
These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d).
The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases.
The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors.
In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy.
Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness.
In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.
File S1 Types of inhibitions with Ki (Compounds 3a-3d).
(DOC) | 1,565 | At what temperatures was the assay completed? | {
"answer_start": [
9753
],
"text": [
"32uC, 37uC, 42uC"
]
} | 3,042 |
864 | Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/
SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b
Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.
Date: 2013-08-05
DOI: 10.1371/journal.pone.0069982
License: cc-by
Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).
Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms.
The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] .
Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] .
Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents.
The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform.
The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools.
N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php
The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads.
Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1-
The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation,
Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation,
The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme.
The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain.
For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.
After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times.
Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] .
Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain.
Type of inhibition Ki (mM) IC 50 (mM)
Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases.
These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d).
The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases.
The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors.
In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy.
Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness.
In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.
File S1 Types of inhibitions with Ki (Compounds 3a-3d).
(DOC) | 1,565 | What criteria sets the guideline for drug-like properties? | {
"answer_start": [
6881
],
"text": [
"Lipinski's ''Rule of five''"
]
} | 3,043 |
865 | Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734177/
SHA: ee8483f8f2cc5fe38be4e565eae3af9d0bb8220b
Authors: Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.
Date: 2013-08-05
DOI: 10.1371/journal.pone.0069982
License: cc-by
Abstract: Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K(i) and IC(50) values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).
Text: Cysteine-protease inhibitors (CPI) have gained considerable attention over the last couple of decades and many classes of compounds are currently in human clinical trials for a number of diseases. Interest in papain family cysteine proteases as chemotherapeutic targets is derived from the recognition that they are critical to the life cycle or pathogenicity of many microorganisms. The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms.
The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] .
Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] .
Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view. They have been shown to possess a broad range of useful pharmacological activities [12] including antigastric, antisecretory, local anesthetic, antiviral, antianxiety, antibacterial, antifungal, antihelminthic, antiprotozoal, anticonvulsant, gastrointestinal, antiulcer (Zolmidine), anxiolytic (Alpidem), hypnotic (Zolpidem) and immunomodulatory [13] . The nature and the position of the substituents on the pyridinic moiety influence these pharmacological activities. These imidazopyridine heterocyclic structures form part of the skeleton of natural alkaloids, neuromuscular blocking agents [14] , reversible inhibitors of the H + , K + -ATPase enzymes with a potent antisecretory activity, and are known to be sedative hypnotics of the nervous system [15] . In this study, we have proposed kinetically and thermodynamically characterized 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as a potent and novel cysteine protease inhibitors which also acts as antibacterial agents.
The crystal structure of papain was extracted from Protein Data Bank (PDB code: 1PE6) [16] . All the water molecules and heteroatoms were removed and hydrogen atoms were added to the protein. CharMm forcefield [17] was applied and the structure was subjected to energy minimization for 1000 steps using steepest descent method. The chemical structures of all the synthesized compounds were generated using chemdraw and were subsequently converted into 3D format using CORINA. A series of docking experiments were carried out with all the designed 1substituted pyridylimidazo[1,5-a]pyridine derivatives against papain using AutoDock Tools 4.0 [18] for possible cysteine-protease inhibitory activities. The compounds were selected on the basis of their binding energies and those reflecting good binding affinity were further analyzed on in silico platform. As a parameter for the molecular docking, the Lamarckian genetic algorithm, a combination between the genetic algorithm and the local search Pseudo-Solis and Wets algorithm, was employed. A grid box of 60660660 Å was generated around active site of papain making sure those inhibitors can freely rotate inside the grid. The number of docking runs was set to 10. Each docking was repeated five times, having in the end a total of 50 docking runs, to check the precision of results. The finally obtained docked complexes were subsequently visualized using PyMol [19] . The work was further authenticated in the wet lab after its detailed analysis on in silico platform.
The designed derivatives were filtered by Lipinski's ''Rule of five'' that sets the criteria for drug-like properties. Drug likeness is a property that is most often used to characterize novel lead compounds [20] . According to this rule, poor absorption is expected if MW .500, log P.5, hydrogen bond donors .5, and hydrogen bond acceptors .10 [21] . In silico absorption, distribution, metabolism and excretion (ADME) properties of these derivatives were also predicted using following online bioinformatics tools.
N http://www.organic-chemistry.org. N http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal. py? Form = admetox N https://secure.chemsilico.com/pages/submit.php
The above study gave us an idea about the existence of possible mutagenic and tumorigenic properties in synthesized compounds. The result obtained helped us to screen out the synthesized compounds for their further usage as potent leads.
Based on the results of docking studies, ten derivatives of 1pyridylimidazo[1,5-a]pyridine were synthesized according to Siddiqui et al., 2006 [22] which are named as follows: 1-
The capacity of the 1-pyridylimidazo[1,5-a]pyridine derivatives to inhibit cysteine proteases was tested using papain as the model enzyme. The proteolytic activity of the reaction mixtures was determined using Bz-DL-Arg-pNA as the chromogenic substrate [23] . To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies. The kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant K i was determined by Dixon method [24] and also by the Lineweaver-Burk equation. The K m value was calculated from the double-reciprocal equation by fitting the data into the computer software Origin 6.1. The Lineweaver-Burk plot was used to determine the types of inhibition. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate, and the average value was considered throughout this work. Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation,
Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation,
The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme.
The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] . In brief, the bacterial strains (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii)) were grown and diluted to 2610 5 colony-forming units (CFU)/ml in sodium phosphate buffer (SPB) containing 0.03% Luria-Bertani (LB) broth. The synthesized derivatives were dissolved in DMSO and their serial dilution was performed in 50 mL of LB medium in 96-well microtitre plate to achieve the required concentrations (0.1-10 mg/ml) with bacterial inoculums (5610 4 CFU per well). DMSO was taken as negative control and Ceftriaxone and clotrimazole were taken as positive control. After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain.
For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.
After having determined the MICs, bacterial strains from the wells of the microtitre plate with no visible bacterial growth were removed for serial sub cultivation of 2 ml into another new microtitre plate containing 100 ml of broth per well and further incubated for 24 h. The lowest concentration with no visible growth was defined as MBC [28], indicating 99.5% killing of the original inoculum. The absorbance of each well was measured at a wavelength of 620 nm by Microplate Manager 4.0 (Bio-Rad laboratories) and compared with a blank. Solvent (DMSO) was used as a negative control. Three replicates were done for each compound and experiment was repeated two times.
Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] .
Turk et al. have proposed, on the basis of kinetic and structural studies, that papain has seven subsites at the active site but only five subsites are important which can bind to an amino acid residue of the substrate [31] . A variety of intermediates are generated when papain reacts with substrate or an inhibitor [2] . Like serine proteases, cysteine proteases tend to have relatively shallow, solvent-exposed active sites that can accommodate short substrate/inhibitor segments of protein loops (e.g. from endogenous inhibitors such as cystatins) or strands. The inhibitor Table 3 . Name, Structure, IC50 & K i of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives against cysteine protease papain.
Type of inhibition Ki (mM) IC 50 (mM)
Non-Competitive 13.7 13.4 compound bound to protease with a combination of hydrogen bonds and hydrophobic interactions. As a part of our investigation in developing novel and efficient cysteine protease inhibitors, ten 1-substituted pyridylimidazo [1,5a] pyridine derivatives (3a-j) were primarily designed and screened on the basis of their docking energies against papain to elucidate their possible mode of action. It was found that these compounds were specific inhibitors of cysteine protease, papain and didn't show inhibition against other types of proteases like serine, aspartic or metalloproteases. They are specific for CA clan of cysteine protease and didn't show any significant inhibition against other clans of cysteine proteases.
These new compounds were devised based on the knowledge of ability of a protein to alter its conformation to accommodate a binding ligand and enabled us to directly compare the relative positions of the residue in the binding pocket. Molecular docking study provided the structural insight into the binding of these compounds (3a-j) (Figure 1 ) within the active site of papain which mainly consist of a catalytic triad of Cys 25, His 159 and Asp 175 [32] . Moreover, role of other residues present in the active site of papain, playing important role in the accommodation of compounds have also been revealed. Initially, docking was performed with all the designed compounds (3a-j) against papain, a known cysteine protease enzyme and in this context, we observed very interesting results where our proposed inhibitors (3a-j) take advantage of aromatic and hydrophilic residues by making a variety of interactions with target enzyme. Although, compounds 3e-j gave significant results when docked with papain but during evaluation of antibacterial properties in wet lab experiments, they gave insignificant results (data not shown). Therefore, only four compounds were considered for discussion and further experiments like kinetic and thermodynamic studies to characterize these compounds as potent pro-inhibitors, were performed (3a-d).
The findings of the above study have shown that the molecular interactions between the compounds 3a-d and papain were very similar to the interactions observed for E-64c, a derivative of naturally occurring epoxide inhibitor (E-64c) (Figure 1 ) of cysteine proteases [31, 32] , with papain; especially with regard to the hydrogen bonding and hydrophobic interactions of the ligands with conserved residues in the catalytic binding site (Figure 2 A-D). Several papain residues participated in hydrophobic interactions with compounds 3a-d, including Gln19, Cys25, Gly66 and Asp158. The pyridine moieties of compounds 3a-d interact with S2 site of papain which includes (Tyr61, Asn64, Gly65 & Tyr67) amino acids (Figure 2 A-D) . The active site residues that were found to be key player in the interaction of compounds within the active site (mostly through hydrophobic interactions) were Cys25, Tyr61, His159 and Trp177, while Trp177, Gln19 were found to me making hydrogen bonds only with compound 3a. Besides this many other residues were also found to be actively involved ( Table 1) . Furthermore, the binding energies for the compound 3a, 3b, 3c and 3d with papain were found to be 26.12, 25.76, 26.84 and 25.62 Kcal/mol respectively, which were in great agreement with our wet lab experiments; shall be discussed later ( Table 1) . This confirmed the accuracy of our docking protocol. Since, the binding energy is a direct measure of strength of interaction and our compounds 3a-d showed stronger binding within the active site of papain in comparison to the inhibitor E-64c (DG: 24.04 Kcal/mol), therefore, the results suggest that these 1-substituted pyridylimidazo[1,5-a]pyridine derivatives (3ad) could be potent inhibitors of papain like cysteine proteases.
The in silico interaction of compounds 3a-d with papain, which were observed as discussed above, was validated with wet lab Table 5 . Prediction of antibacterial compounds as drugs (http://www.organic-chemistry.org). Table 2) . Interestingly, the observed in silico binding energies for the compounds 3a-d against papain were found to be in great agreement (standard error 62 Kcal/mol) with the value of free energy of binding (DG) observed during thermodynamics studies ( Table 1 and 2) . Similarly, enthalpy change (DH) of the binding was negative whereas entropy (DS) change of the binding was positive which indicated the exothermic and entropically driven nature of binding. This pattern of temperature dependence is characteristic of hydrophobic interaction [33] . As discussed earlier that all the compounds (3a-d) were found to interact with the active site residues of papain through hydrophobic interactions at most instances during in silico studies, the same was observed by the analysis of Van't Hoff plots for all the proposed inhibitors at three different temperatures (32uC, 37uC and 42uC) in wet lab experiments ( Figure 3) . This proves the importance of these types of interactions in the positioning of compounds within the active site. Hence, thermodynamics as well as in silico study reveals that hydrophobic interactions favor binding of these proposed inhibitors with papain like cysteine proteases. Further wet lab results proposed the non competitive interaction of compounds (3a, 3c & 3d) with papain except for compound 3b which showed competitive interaction. In sum up, the above results of molecular docking studies and thermodynamic analysis of compounds 3a-d with papain showed that these compounds have the potential to be novel and unique cysteine protease inhibitors.
In the current study, the cysteine protease inhibitory activity of synthesized derivatives of 1-substituted pyridylimidazo[1,5-a] pyridine (3a-d)) was also performed against papain and the inhibition constants (K i ) for the above said enzyme were observed to be 13.70, 23.20, 90.00 and 99.30 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values. Compounds 3c & 3d are having much difference in their IC50 values (3b-21.17 mM and 3c-94.5 mM) and they showed exact pattern for their antibacterial activity for all microbes except for E. coli at one instance. Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy.
Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference. So, Clotrimazole (1-[(2-chlorophenyl)(diphenyl)methyl]-1H-imidazole), an imidazole derivatives and Ceftriaxone (third-generation cephalosporin antibiotic with broad spectrum activity against Gram-positive and Gram-negative bacteria) have been used as positive control whereas DMSO has been used as negative control. All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity. The distribution of calculated logP (cLogP) values of a majority of drugs in the market is in the range of zero to five. All the compounds studied except 3d, showed good agreement with the criteria laid down for the prediction of a compound to be a potential drug ( Table 5 ). All the compounds do not show any threat against toxicity risk assessment except compound 3d which showed threat as tumorogenic effect due to the presence of isobutyl group. Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness.
In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.
File S1 Types of inhibitions with Ki (Compounds 3a-3d).
(DOC) | 1,565 | What could be novel candidates as potent inhibitors of papain like cysteine proteases in resistant microorganisms? | {
"answer_start": [
22962
],
"text": [
"1-substituted pyridylimidazo[1,5-a]pyridine derivatives"
]
} | 3,044 |
866 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What causes acute respiratory illness in young children? | {
"answer_start": [
427
],
"text": [
"Human metapneumovirus (HMPV)"
]
} | 4,059 |
867 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What is the molecular structure of the Human metapneumovirus (HMPV)? | {
"answer_start": [
1725
],
"text": [
"single-stranded RNA virus"
]
} | 4,060 |
868 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What virus is closely related to the human respiratory syncytial virus (RSV)? | {
"answer_start": [
1691
],
"text": [
"Human metapneumovirus (HMPV)"
]
} | 4,061 |
869 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What diseases are caused by HMPV? | {
"answer_start": [
1911
],
"text": [
"mild upper respiratory infection to bronchiolitis and pneumonia"
]
} | 4,062 |
870 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | How large is the HMPV genome? | {
"answer_start": [
2108
],
"text": [
"13 kb"
]
} | 4,063 |
871 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | How many open reading frames are in the HMPV genome? | {
"answer_start": [
2128
],
"text": [
"eight"
]
} | 4,064 |
872 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What are the two major genotypes of HMPV? | {
"answer_start": [
2486
],
"text": [
"A and B"
]
} | 4,065 |
873 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What is the most common subgroup of HMPV? | {
"answer_start": [
2566
],
"text": [
"HMPV A2"
]
} | 4,066 |
874 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | Who accounted for 44% of HMPV positive cases in Kenya between 2007 and 2011? | {
"answer_start": [
3234
],
"text": [
"children under 6 months of age"
]
} | 4,067 |
875 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What does this study describe? | {
"answer_start": [
5474
],
"text": [
"a whole genome sequencing (WGS) approach for HMPV"
]
} | 4,068 |
876 | Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941262/
SHA: f5ae3f66face323615df39d838e056ab5fcc98df
Authors: Kamau, Everlyn; Oketch, John W.; de Laurent, Zaydah R.; Phan, My V. T.; Agoti, Charles N.; Nokes, D. James; Cotten, Matthew
Date: 2020-01-02
DOI: 10.1186/s12864-019-6400-z
License: cc-by
Abstract: BACKGROUND: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. RESULTS: We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. CONCLUSION: This is the first genomic characterization of HMPV genomes from African patients.
Text: Human metapneumovirus (HMPV) is a single-stranded RNA virus in the family Paramyxoviridae and closely related to human respiratory syncytial virus (RSV) [1] . HMPV causes respiratory disease similar to RSV, ranging from mild upper respiratory infection to bronchiolitis and pneumonia [2] . HMPV infections are seasonal and coinfection with other respiratory pathogens is common [1] . The HMPV genome is approximately 13 kb and comprises eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), transcription enhancer protein (M2), small hydrophobic protein (SH), attachment glycoprotein (G), and large polymerase protein (L) [3] . The membrane glycoproteins F and G sequences are used to define two major genotypes or groups, A and B, which are further classified into four subgroups (A1, A2, B1, and B2). HMPV A2, the most frequently observed subgroup, is further divided into two proposed sub-lineages (A2a and A2b) [3] .
HMPV is reported to have an important contribution to acute respiratory infections (ARI) in Africa. For instance, HMPV-associated hospitalization was estimated at 6.5 per 1000 person years in infants in Soweto, South Africa [4] ; at 4% in hospitalized children with severe ARI during a 2-year period in Cameroon [5] ; and in rural western Kenya, incidence of HMPV associated with ARI cases in outpatient clinic visits was estimated at 0.43 per 100 person-years among outpatients [6] . In Kilifi coastal Kenya, between January 2007 to December 2011, children under 6 months of age accounted for 44% of HMPV positive cases, while 74% were children under 1 year, and 1.3% (2/160) were children > 36 months [7] . In Dadaab and Kakuma refugee camps in Kenya, HMPV was detected in 5.7% hospitalizations, and virus-positive crude hospitalization rate (per 1000 children < 5 years old) was 4 for HMPV [8] . In Mali, contribution of HMPV to pneumonia had a population attributable fraction of 9% (95% CI: 7-11%) [9] ; while in Morocco [10] , 8 .9% of children < 5 years admitted with severe pneumonia were infected with HMPV. HMPV prevalence and incidence elsewhere globally, is indicated in Additional file 4: Table S1 . Of note is that the variations in incidence rates could be attributed to study population, seasonality and even detection methods. Nonetheless, genomic epidemiology of HMPV in Africa is inadequately reported, and comparison of genetic similarity and differences between African and global strains is not documented.
Genome sequences provide valuable resources for characterizing viral evolution and disease epidemiology, and for identifying transmission events and outbreaks, which is not always possible with sub-genomic fragments [11] [12] [13] . The increased number of phylogenetically informative variant sites obtained from full genomes may allow better linking of cases and aid public health interventions in real time during epidemics [14, 15] . PCR approaches for targeted whole genome sequencing, in contrast to random amplification, can preferentially amplify the target virus over host or environmental nucleic acids [16, 17] potentially focusing sequencing on the virus of interest. To date, the largest dataset of HMPV whole genomes (n = 61) sequenced from any tropical country is from three Peruvian cities, Lima, Piura and Iquitos [18] . In Africa, apart from one metapneumovirus genome identified from a wild mountain gorilla in Rwanda (GenBank accession number HM197719), there are no HMPV genomes reported according to the NIAID Virus Pathogen Database and Analysis Resource (ViPR, http://www.viprbrc. org/, accessed April 30, 2019). This has led to limited understanding of the genetic and genomic diversity of HMPV in the continent.
This work describes a whole genome sequencing (WGS) approach for HMPV from a small number of HMPV positive clinical samples collected at Kilifi County Hospital in Kilifi, Kenya and University Teaching Hospital in Lusaka, Zambia. The genomes were generated by sequencing overlapping PCR amplicons spanning the entire genome. These are the first reported complete genome sequences of locally circulating HMPV strains obtained directly from clinical samples in Africa. We also combined the new genomes with publicly available sequences to examine patterns in global HMPV genetic diversity.
Whole genome sequencing was successful for all 5 clinical samples that were attempted. A single genomic sequence was obtained from each sample, and the length of the 5 new HMPV genomes ranged from 13,097 to 13, 134 nt (> 95% length coverage). Sequencing and data assembly parameters, including coverage depth are shown in Table 1 .
Sequence annotation of the full-length genomes using Geneious R8.1.5 (https://www.geneious.com) identified the expected eight coding ORFs and non-coding genomic regions. The overall nucleotide identity (i.e., identical sites averaging over all sequence pairs and excluding positions containing gaps) between all 143 genome sequences analyzed (5 new genomes plus 138 from ViPR) was 58.2%. Nucleotide sequence identity was 71.3% within HMPV-A and 80% within HMPV-B. Intrasubgroup, A1, A2, B1 and B2 genomes shared 92.1% (10 sequences), 76.8% (88 sequences), 91% (24 sequences) and 89.6% (21 sequences) amino acid sequence identity.
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .
A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 . | 1,591 | What was the difference in the group A and B genomes? | {
"answer_start": [
7202
],
"text": [
"The length of the F-M2 intergenic region"
]
} | 4,069 |
877 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | Which are the most abundant biological entities on Earth? | {
"answer_start": [
574
],
"text": [
"Viruses "
]
} | 1,175 |
878 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What contributed to a large part of mammalian genomic sequence? | {
"answer_start": [
731
],
"text": [
"Retroviruses "
]
} | 1,176 |
879 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What were the earliest replicating entities that fulfill several criteria for life? | {
"answer_start": [
1032
],
"text": [
"ribozymes or viroids"
]
} | 1,177 |
880 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What are some examples of autonomous bacteria that lost their independence and became intracellular parasites or endosymbionts? | {
"answer_start": [
1444
],
"text": [
"mitochondria, chloroplasts, Rickettsia "
]
} | 1,178 |
881 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What entities with no genes satisfy the criteria for life? | {
"answer_start": [
2433
],
"text": [
"ribozymes, catalytic RNAs closely related to viroids"
]
} | 1,179 |
882 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | Which group of RNA quasispecies satisfy criteria for life? | {
"answer_start": [
2682
],
"text": [
" catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 ("
]
} | 1,180 |
883 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | How are the ribozymes able to replicate, join and create peptide bonds? | {
"answer_start": [
2958
],
"text": [
" They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. "
]
} | 1,181 |
884 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | Does RNA replication need polymerase enzymes? | {
"answer_start": [
3512
],
"text": [
"Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes."
]
} | 1,182 |
885 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | How can DNA arise chemically from RNA? | {
"answer_start": [
3619
],
"text": [
"deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase."
]
} | 1,183 |
886 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What do ribozymes consist of? | {
"answer_start": [
3946
],
"text": [
"circular single-stranded RNAs"
]
} | 1,184 |
887 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What do ribozymes lack? | {
"answer_start": [
4002
],
"text": [
"the genetic triplet code and do not encode proteins"
]
} | 1,185 |
888 | Viruses and Evolution – Viruses First? A Personal Perspective
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/
SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1
Authors: Moelling, Karin; Broecker, Felix
Date: 2019-03-19
DOI: 10.3389/fmicb.2019.00523
License: cc-by
Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.”
Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities?
There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) .
The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase.
An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) .
tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) .
Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) .
These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) .
It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome.
RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black.
well as during retroviral replication, proving a requirement of RNA (Flint, 2015) .
The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?
According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) .
Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element.
Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) .
From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs.
information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) .
The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle.
Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost.
Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation.
The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes.
There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist?
In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems.
In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first."
ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity.
The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.
These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ).
An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment.
The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) .
To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.
The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) .
The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) .
Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below).
Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) .
The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) .
The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) .
There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions.
One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations.
ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy.
Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) .
A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses?
These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) .
Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred.
Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria.
Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) .
HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) .
Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) .
Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms.
Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) .
The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) .
Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) .
Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text.
to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion.
One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations?
The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) .
A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know.
Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) .
There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) .
The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts.
Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic?
Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others.
A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) .
These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes.
There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) .
Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species.
Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution.
There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA.
Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution.
The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life.
The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence. | 1,690 | What do ribozymes exhibit? | {
"answer_start": [
4077
],
"text": [
"structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules."
]
} | 1,186 |
889 | Potential Maternal and Infant Outcomes from (Wuhan) Coronavirus 2019-nCoV Infecting Pregnant Women: Lessons from SARS, MERS, and Other Human Coronavirus Infections
https://doi.org/10.3390/v12020194
SHA: 779c1b5cb3afe3d50219aa2af791014a22eb355a
Authors: Schwartz, David A.; Graham, Ashley L.
Date: 2020
DOI: 10.3390/v12020194
License: cc-by
Abstract: In early December 2019 a cluster of cases of pneumonia of unknown cause was identified in Wuhan, a city of 11 million persons in the People’s Republic of China. Further investigation revealed these cases to result from infection with a newly identified coronavirus, termed the 2019-nCoV. The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. Similar to such other coronaviruses as those causing the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the new coronavirus was reported to spread via natural aerosols from human-to-human. In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. Unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-nCoV epidemic. In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants. Recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-nCoV vaccines.
Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses. They have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] .
In humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] . Between 8 and 18 December 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market. This market, termed a "wet" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation. As the disease was spreading through secondary and tertiary cases, the World Health Organization (WHO) China Country Office was informed on 31 December 2019 of the occurrence of these cases of pneumonia of unknown etiology. During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO. On 7 January 2020 investigators in China identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-nCoV (for 2019 novel coronavirus) [8] . Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. The most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died. During the first weeks of January the infection spread rapidly through China and extended to adjacent countries where cases began to appear-13 January in Thailand, 15 January in Japan, 20 January in the Republic of Korea, and Taiwan and the United States on 21 January [11] . Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan. The new coronavirus continued to spread throughout multiple countries and continents, and by 9 February 2020 the WHO reported 37,251 confirmed cases in China that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 SARS epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] .
Viruses 2020, 12, 194 3 of 16 epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy.
Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy.
Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (PROM) and preterm labor (PTL), intrauterine fetal demise (IUFD), intrauterine growth restriction (IUGR), and neonatal death [14] [15] [16] .
The SARS epidemic began quietly at the turn of the 21st century. In November 2002, a cook in Guangdong Province, China, died from an unidentified illness. He had worked at a restaurant in which meat from wild animals was served. On 27 November 2002 Chinese-language media and internet reports were picked up by Canada's Global Public Health Intelligence Network (GPHIN) that indicated a flu-like illness was occurring in China [25, 26] . Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003. The disease spread to other countries where it primarily infected healthcare workers. One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi. He recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the WHO. He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference. On 12 March 2003, WHO issued a global alert regarding the disease that was occurring primarily among health care workers in Hanoi, Vietnam and Hong Kong. The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong. Approximately 30% of infections occurred in healthcare workers. By the termination of the epidemic the global CFR was 11% [27] .
Although there were relatively few documented cases of SARS occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. However, even if this is the case, there is no doubt that SARS coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] .
The clinical outcomes among pregnant women with SARS in Hong Kong were worse than those occurring in infected women who were not pregnant [32] . Wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed SARS in Hong Kong during the period of 1 February to 31 July 2003. Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress. Among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%).
A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] . There were 3 deaths among the pregnant women with SARS (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (P = 0.006). Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group. Six pregnant women with SARS required admission to the intensive care unit (ICU) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (P = 0.065) and 17.5% ICU admission rate (P = 0.012) in the non-pregnant group.
Maxwell et al. [32] reported 7 pregnant women infected with SARS-CoV who were followed at a designated SARS unit-2 of the 7 died (CFR of 28%), and 4 (57%) required ICU hospitalization and mechanical ventilation. In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV. Two women with SARS recovered and maintained their pregnancy but had infants with IUGR. Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection. The new mothers who had developed SARS were advised not to breastfeed to prevent possible vertical transmission of the virus.
Zhang et al. [34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic. Two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired. All 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (ALT), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. One pregnant woman required intensive care, but all recovered and there were no maternal deaths. The 5 infants were clinically evaluated, and none had evidence of SARS.
Two pregnant women with SARS were reported from the United States. In a detailed case report, Robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV. At 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. Upon returning to the United States she was hospitalized with pneumonia. Obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV. She underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . The placenta was interpreted as being normal. At 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral RNA was detected in specimens tested by reverse transcriptase polymerase chain reaction (RT-PCR). Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay. No clinical specimens (except for cord blood) were available for testing from the infant. The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] . Following her return to the United States, her husband developed the clinical onset of SARS, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays. Her pregnancy continued and was unremarkable except for developing elevated glucose levels. A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. At the time of delivery, the mother's serum samples were positive for antibodies to SARS-CoV, but samples taken of umbilical cord blood and placenta were negative. Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies. Specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus RNA by RT-PCR. Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA.
From Canada, Yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. She had acquired SARS from contact with an infected family member. Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. She had a normal labor and delivery and her newborn girl had no evidence of infection.
In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] . None of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise.
Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred.
In the only reported study of the placental pathology of mothers with SARS, Ng et al. [41] reported the findings from 7 pregnant women infected with SARS-CoV. In the case of 2 women who were convalescing from SARS-CoV infection during the 1st trimester of pregnancy, the placentas were found to be normal. Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. In the placentas of 2 women who were convalescing from SARS-CoV infection in the 3rd trimester of pregnancy the placentas were highly abnormal. They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. These 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed IUGR. It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas.
Similar to other coronavirus infections, SARS-CoV is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] . Environmental factors had an important role when it was discovered that during the Amoy Gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, SARS-CoV was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection.
In order to address the safety issues for the obstetrical management and delivery of pregnant women with SARS, guidelines were prepared by the Canadian Task Force on Preventive Health Care and the Society of Obstetricians and Gynaecologists of Canada [45] . These recommendations include:
1.
"All hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for SARS or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit.
At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS.
Upon arrival in the labour triage unit, pregnant patients with suspected and probable SARS should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation.
If possible, labour and delivery (including operative delivery or Caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5.
Either regional or general anaesthesia may be appropriate for delivery of patients with SARS.
Neonates of mothers with SARS should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. The mother should not breastfeed during this period. 7.
A multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of SARS management protocols. 8.
Staff caring for pregnant SARS patients should not care for other pregnant patients. Staff caring for pregnant SARS patients should be actively monitored for fever and other symptoms of SARS. Such individuals should not work in the presence of any SARS symptoms within 10 days of exposure to a SARS patient. 9.
All health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the SARS virus. 10. Regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with SARS or similar illnesses."
Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . In the 8 years since then, there have been more than 2494 confirmed cases of MERS resulting in upwards of 858 deaths globally [46] . While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] . To date, all known cases of MERS can be linked to travel or residence in countries along the Arabian Peninsula-that is, Bahrain; Iraq; Iran; Israel, the West Bank, and Gaza; Jordan; Kuwait; Lebanon; Oman; Qatar, Saudi Arabia; Syria; the United Arab Emirates (UAE); and Yemen [48] . The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] . The index case in this outbreak reportedly returned from the Arabian Peninsula just prior to onset of illness [50] .
MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . Nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of MERS [1] . Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] . Like other coronaviruses, MERS-CoV can be spread through person-to-person contact, likely via infected respiratory secretions [48] . Transmission dynamics, however, are otherwise poorly understood [1] . Bats are believed to be the natural reservoir of MERS-CoV, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] .
There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] . Researchers have not yet discovered ongoing transmission of MERS-CoV within communities outside of health care settings.
The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death. Most patients with MERS develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . MERS progresses much more rapidly to respiratory failure and has a higher case fatality rate than SARS [1] . Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . The overall CFR of MERS is approximately 34.4% [46] . Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. As a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy.
Limited data exists on the prevalence and clinical features of MERS during pregnancy, birth, and the postnatal period. It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] . Pregnant women infected with SARS-CoV, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that MERS-CoV could also lead to severe clinical outcomes in pregnancy. To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes.
Between November 2012 and February 2016, there were 1308 cases of MERS reported by the Saudi Arabia Ministry of Health (MoH). Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al. [47] , and all resulted in adverse outcomes. Patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. All 5 cases received intensive care. Two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection. The authors of the retrospectives study concede that unreported cases of MERS in pregnancy are likely due to lack of routine pregnancy testing [47] . They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk. Additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to MERS-CoV in healthcare settings.
In a separate case report of MERS occurring in pregnancy, Alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. Following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (ICU) and later recovered. The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. In contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of MERS-CoV exposure, her young age, the use of steroids, and differences in immune response.
Alfaraj et al. [61] described 2 cases of maternal infection with MERS-CoV at the Prince Mohammed Bin Abdulaziz Hospital (PMAH) in Saudi Arabia. Maternal infection in both cases was confirmed by nasopharyngeal swab testing by RT-PCR. One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. The second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak. Both patients later tested negative for MERS-CoV and were subsequently discharged. The younger patient delivered a healthy, full-term infant. The status of the other delivery is unknown. Neither fetus was tested for MERS-CoV.
According to Payne et al. [62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV. The mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. On the 7th day of symptoms, she had a fetal death. The mother was confirmed to have antibody to MERS-CoV, and she self-reported having had unprotected contact with family members who later tested positive for the virus. This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV.
On 24 November 2013, a 32-year-old pregnant woman in the United Arab Emirates (UAE) developed ARDS following admission to the ICU after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range. The next day, RT-PCR evaluation revealed that the mother was positive for MERS-CoV. Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. While the outcome for this mother was fatal, Malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . More research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] .
Outside of the Middle East the only confirmed case of MERS in pregnancy occurred in 2015 in South Korea. Jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having MERS. Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. Subsequent testing of the infant's blood did not detect any IgG, IgM, or IgA antibodies to MERS-CoV.
The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. The source of infection in 2 of the cases was attributed to contact with family members who tested positive for MERS-CoV, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). Six patients required intensive care and 3 died. Of those who died, 2 were exposed to MERS-CoV in the 3rd trimester, and 1 was exposed during the 2nd trimester. The infant death rate for all 11 cases was 27%. Fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. According to Alfaraj et al. [61] , the CFR for the 11 infected women-also 27%-was not statistically different from the overall CFR of MERS in the general population (35%) (P = 0.75). Only 1 case resulted in both maternal and fetal death.
Similar to SARS in pregnancy, more research is needed to understand the pathogenesis and epidemiology of MERS in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. As such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] .
MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . Since 2013, the Saudi Arabia MoH has recommended that pregnant women postpone travel to Saudi Arabia for the Hajj and Umrah [47] . To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. Pregnant women who present with symptoms of pneumonia, influenza-like illness (ILI), or sepsis on the Arabian Peninsula may also benefit from MERS-CoV screening to expedite early diagnosis and improve disease management [60] .
While multiple agents have been used to treat MERS, none have been tested in large clinical studies. Available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks.
Due to a gap in research on the treatment of MERS in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. For example, during the 2003 SARS outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] .
The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold. In order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, Gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. These specimens were evaluated for the presence of HCoV 229E, OC-43, NL63 and HKU 1 using RT-PCR methodology. Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads. Human coronaviruses were identified in 12 samples (HCoV 229E: 11; HKU 1 : 1) from 7 mother-child pairs. In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. There were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus.
It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. There are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to SARS-CoV, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] .
It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV. In examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . Excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] .
On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus. The Chief Executive Officer of CEPI, Richard Hatchett, said [72] :
"Given the rapid global spread of the nCoV-2019 virus the world needs to act quickly and in unity to tackle this disease. Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development."
The novel coronavirus is the first epidemic disease to emerge since the formation of CEPI in Davos in 2017. CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. In May 2017, WHO released the Target Product Profile (TPP) for MERS-CoV vaccines, following the prioritization of MERS-CoV as one of eight priority pathogens for prevention of epidemics [73] . CEPI and partners aim to use existing platforms-that is, the existing "backbone" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates. Following the WHO declaration on 30 January that the current 2019-nCoV outbreak is a public health emergency of international concern (PHEIC), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease.
A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? As of the PHEIC declaration, 12 groups have announced that they are developing new vaccines against 2019-nCoV and seven others announced initiatives to develop new therapies [74] . Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. To date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rVSV-ZEBOV vaccine through 3 Ebola virus epidemics serves as a recent example [69] [70] [71] . Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses.
On 5 February 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in Wuhan had tested positive for 2019-nCoV at the Wuhan Children's Hospital in Hubei Province 30 hours following its birth. According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus. Reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , "This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission"
The hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020. Following its birth, the infant's nanny was diagnosed with 2019-nCoV, and the mother was diagnosed days later [76] . On 29 January the baby began to develop symptoms. According to Dr. Zeng Lingkong [76] , "Whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected"
In considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. In some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. Based upon past experience with pregnant women who developed MERS and SARS, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , "It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat," "It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing."
And according to Dr. Paul Hunter, Professor of Medicine at the University of East Anglia [79] , "As far as I am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth."
There is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, SARS and MERS. These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. Both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death. Unlike some viral infections, notably Ebola virus [70] and Zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either SARS or MERS. It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. However, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-nCoV. Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. This research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks. | 2,551 | What are coronaviruses? | {
"answer_start": [
1930
],
"text": [
"s are spherical, enveloped, and the largest of positive-strand RNA v"
]
} | 2,199 |
890 | Potential Maternal and Infant Outcomes from (Wuhan) Coronavirus 2019-nCoV Infecting Pregnant Women: Lessons from SARS, MERS, and Other Human Coronavirus Infections
https://doi.org/10.3390/v12020194
SHA: 779c1b5cb3afe3d50219aa2af791014a22eb355a
Authors: Schwartz, David A.; Graham, Ashley L.
Date: 2020
DOI: 10.3390/v12020194
License: cc-by
Abstract: In early December 2019 a cluster of cases of pneumonia of unknown cause was identified in Wuhan, a city of 11 million persons in the People’s Republic of China. Further investigation revealed these cases to result from infection with a newly identified coronavirus, termed the 2019-nCoV. The infection moved rapidly through China, spread to Thailand and Japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. Similar to such other coronaviruses as those causing the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), the new coronavirus was reported to spread via natural aerosols from human-to-human. In the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. Unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-nCoV epidemic. In order to assess the potential of the Wuhan 2019-nCoV to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of SARS, MERS, and other coronavirus infections on pregnant women and their infants. Recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-nCoV vaccines.
Text: Coronaviruses are spherical, enveloped, and the largest of positive-strand RNA viruses. They have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. Belonging to the order Nidovirales, family Coronaviridae, and the subfamily Orthocoronaviridae there are four genera of coronaviruses-Alphacoronavirus, Betacoronavirus, Deltacorona virus, and Gammacoronavirus [1] [2] [3] [4] .
In humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. However, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)-can produce severe lower respiratory In the beginning of December 2019, a cluster of persons with a pneumonia of unknown cause was identified in Wuhan, the capital of Hubei Province and a large city of approximately 11 million persons located in the central region of the People's Republic of China [7, 8] . Between 8 and 18 December 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. The outbreak was initially believed to be linked to the Wuhan Huanan (South China) Seafood Wholesale Market. This market, termed a "wet" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . On 31 December 2019, the Chinese Center for Disease Control and Prevention (China CDC) sent a rapid response team to Hubei to work alongside health personnel from the provincial and Wuhan city health departments to conduct an epidemiologic investigation. As the disease was spreading through secondary and tertiary cases, the World Health Organization (WHO) China Country Office was informed on 31 December 2019 of the occurrence of these cases of pneumonia of unknown etiology. During the period from 31 December 2019 to 3 January 2020, 44 patients with pneumonia of unknown etiology were reported by the Chinese authorities to the WHO. On 7 January 2020 investigators in China identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-nCoV (for 2019 novel coronavirus) [8] . Analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-nCoV infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. The most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . Among these 41 initial cases of 2019-nCoV infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ARDS), 13 (32%) required intensive care and 6 (15%) died. During the first weeks of January the infection spread rapidly through China and extended to adjacent countries where cases began to appear-13 January in Thailand, 15 January in Japan, 20 January in the Republic of Korea, and Taiwan and the United States on 21 January [11] . Infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of Wuhan. The new coronavirus continued to spread throughout multiple countries and continents, and by 9 February 2020 the WHO reported 37,251 confirmed cases in China that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 SARS epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] .
Viruses 2020, 12, 194 3 of 16 epidemic. An additional 307 cases of 2019-nCoV infection have occurred among 24 other countries outside of China [12] . (Figure 1 ) At the meeting of the Emergency Committee of the WHO on 30 January, the novel coronavirus 2019 epidemic was declared a Public Health Emergency of International Concern (PHEIC) [11, 13] . This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy.
Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include This newly recognized coronavirus, producing a disease that has been termed COVID-19, is rapidly spreading throughout China, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. It is certain that 2019-nCoV will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with SARS-CoV and MERS-CoV. In order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy.
Pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. It is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . In one study pneumonia was the 3rd most common cause of indirect maternal death [17] . Approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . Although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . As with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . This has been evident historically during previous epidemics. The case fatality rate (CFR) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . During the 1957-1958 Asian flu epidemic, 10% of all deaths occurred in pregnant women, and their CFR was twice as high as that of infected women who were not pregnant [24] . The most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (PROM) and preterm labor (PTL), intrauterine fetal demise (IUFD), intrauterine growth restriction (IUGR), and neonatal death [14] [15] [16] .
The SARS epidemic began quietly at the turn of the 21st century. In November 2002, a cook in Guangdong Province, China, died from an unidentified illness. He had worked at a restaurant in which meat from wild animals was served. On 27 November 2002 Chinese-language media and internet reports were picked up by Canada's Global Public Health Intelligence Network (GPHIN) that indicated a flu-like illness was occurring in China [25, 26] . Unfortunately, the reports were not translated, and China failed to report the occurrence of this illness to the World Health Organization (WHO) until February 2003. The disease spread to other countries where it primarily infected healthcare workers. One of these was Dr. Carlo Urbani, a WHO physician investigating a patient with the new disease in Hanoi. He recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the WHO. He contracted the SARS-CoV while there, became febrile and later died after traveling to Thailand to attend a conference. On 12 March 2003, WHO issued a global alert regarding the disease that was occurring primarily among health care workers in Hanoi, Vietnam and Hong Kong. The disease continued to spread, and by 31 July 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland China and Hong Kong. Approximately 30% of infections occurred in healthcare workers. By the termination of the epidemic the global CFR was 11% [27] .
Although there were relatively few documented cases of SARS occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. In reviewing these reports describing pregnant women with SARS in China it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. However, even if this is the case, there is no doubt that SARS coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . Martha Anker, an expert in statistics formerly with the WHO and the University of Massachusetts, estimated that more than 100 cases of SARS-CoV infection occurred in pregnant women, which warrants closer inspection [27] .
The clinical outcomes among pregnant women with SARS in Hong Kong were worse than those occurring in infected women who were not pregnant [32] . Wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed SARS in Hong Kong during the period of 1 February to 31 July 2003. Four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by SARS-related acute respiratory distress. Among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%).
A case-control study to determine the effects of SARS on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the Princess Margaret Hospital in Hong Kong [27, 33] . There were 3 deaths among the pregnant women with SARS (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (P = 0.006). Renal failure (P = 0.006) and disseminated intravascular coagulopathy (P = 0.006) developed more frequently in pregnant SARS patients when compared with the non-pregnant SARS group. Six pregnant women with SARS required admission to the intensive care unit (ICU) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (P = 0.065) and 17.5% ICU admission rate (P = 0.012) in the non-pregnant group.
Maxwell et al. [32] reported 7 pregnant women infected with SARS-CoV who were followed at a designated SARS unit-2 of the 7 died (CFR of 28%), and 4 (57%) required ICU hospitalization and mechanical ventilation. In contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with SARS-CoV. Two women with SARS recovered and maintained their pregnancy but had infants with IUGR. Among the live newborn infants, none had clinical or laboratory evidence for SARS-CoV infection. The new mothers who had developed SARS were advised not to breastfeed to prevent possible vertical transmission of the virus.
Zhang et al. [34] described SARS-CoV infections in 5 primagravidas from Guangzhou, China at the height of the SARS epidemic. Two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. Two of the pregnant women had hospital-acquired SARS infections, and the other 3 were community-acquired. All 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (ALT), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. One pregnant woman required intensive care, but all recovered and there were no maternal deaths. The 5 infants were clinically evaluated, and none had evidence of SARS.
Two pregnant women with SARS were reported from the United States. In a detailed case report, Robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. While travelling in Hong Kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with SARS-CoV. At 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. Upon returning to the United States she was hospitalized with pneumonia. Obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. Following her discharge home and clinical recovery, she was found to have antibodies to SARS-CoV. She underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . The placenta was interpreted as being normal. At 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral RNA was detected in specimens tested by reverse transcriptase polymerase chain reaction (RT-PCR). Antibodies to SARS-CoV were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (EIA) and indirect immunofluorescence assay. No clinical specimens (except for cord blood) were available for testing from the infant. The second case in the USA occurred in a 38-year-old woman who had travelled to Hong Kong at 7 weeks gestation where she was exposed to SARS-CoV in the same hotel as the aforementioned American woman [37] . Following her return to the United States, her husband developed the clinical onset of SARS, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. Following her hospitalization for SARS she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to SARS-CoV by enzyme immunoassay and immunofluorescent assays. Her pregnancy continued and was unremarkable except for developing elevated glucose levels. A cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. At the time of delivery, the mother's serum samples were positive for antibodies to SARS-CoV, but samples taken of umbilical cord blood and placenta were negative. Breast milk sampled 12 and 30 days after delivery were also negative for SARS-CoV antibodies. Specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus RNA by RT-PCR. Neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral RNA.
From Canada, Yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. She had acquired SARS from contact with an infected family member. Following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. She had a normal labor and delivery and her newborn girl had no evidence of infection.
In a study of 5 liveborn neonates who were delivered to women infected with SARS-CoV during the Hong Kong epidemic, results from multiple tests-including serial RT-PCR assays, viral culture, and paired neonatal serological titers-were negative for SARS-CoV [39] . None of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise.
Fortunately, there were no cases of vertical transmission identified among pregnant women infected with SARS-CoV during the 2002-2003 Asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of SARS have occurred.
In the only reported study of the placental pathology of mothers with SARS, Ng et al. [41] reported the findings from 7 pregnant women infected with SARS-CoV. In the case of 2 women who were convalescing from SARS-CoV infection during the 1st trimester of pregnancy, the placentas were found to be normal. Three placentas were delivered from pregnancies in which the mothers had acute SARS-CoV infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. In the placentas of 2 women who were convalescing from SARS-CoV infection in the 3rd trimester of pregnancy the placentas were highly abnormal. They showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. These 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed IUGR. It is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas.
Similar to other coronavirus infections, SARS-CoV is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . In addition to transmission of SARS-CoV through natural aerosols from infected patients, it was found that in Hong Kong the SARS-CoV could also be transmitted by mechanical aerosols [44] . Environmental factors had an important role when it was discovered that during the Amoy Gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, SARS-CoV was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . Healthcare facilities were also an important source of new SARS infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection.
In order to address the safety issues for the obstetrical management and delivery of pregnant women with SARS, guidelines were prepared by the Canadian Task Force on Preventive Health Care and the Society of Obstetricians and Gynaecologists of Canada [45] . These recommendations include:
1.
"All hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for SARS or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit.
At times of SARS outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for SARS.
Upon arrival in the labour triage unit, pregnant patients with suspected and probable SARS should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. All labour and delivery units caring for suspected and probable SARS should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation.
If possible, labour and delivery (including operative delivery or Caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5.
Either regional or general anaesthesia may be appropriate for delivery of patients with SARS.
Neonates of mothers with SARS should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. The mother should not breastfeed during this period. 7.
A multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of SARS management protocols. 8.
Staff caring for pregnant SARS patients should not care for other pregnant patients. Staff caring for pregnant SARS patients should be actively monitored for fever and other symptoms of SARS. Such individuals should not work in the presence of any SARS symptoms within 10 days of exposure to a SARS patient. 9.
All health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the SARS virus. 10. Regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with SARS or similar illnesses."
Middle East respiratory syndrome (MERS) was first reported in September 2012 in Saudi Arabia, following isolation of MERS-CoV from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . In the 8 years since then, there have been more than 2494 confirmed cases of MERS resulting in upwards of 858 deaths globally [46] . While 27 countries have reported cases of MERS, approximately 80% of confirmed cases originated in Saudi Arabia [47] . To date, all known cases of MERS can be linked to travel or residence in countries along the Arabian Peninsula-that is, Bahrain; Iraq; Iran; Israel, the West Bank, and Gaza; Jordan; Kuwait; Lebanon; Oman; Qatar, Saudi Arabia; Syria; the United Arab Emirates (UAE); and Yemen [48] . The largest documented outbreak outside of this region occurred in 2015 in the Republic of Korea, in which 186 infections occurred, resulting in 38 deaths [49] . The index case in this outbreak reportedly returned from the Arabian Peninsula just prior to onset of illness [50] .
MERS-CoV is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . Nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of MERS [1] . Superspreading events have been recorded in healthcare settings in Jordan, Al Hasa, Jeddah, Abu Dhabi and South Korea [47, [52] [53] [54] [55] . Like other coronaviruses, MERS-CoV can be spread through person-to-person contact, likely via infected respiratory secretions [48] . Transmission dynamics, however, are otherwise poorly understood [1] . Bats are believed to be the natural reservoir of MERS-CoV, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] .
There are no clinical or serological reports of perinatal transmission of MERS, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (RSV) [58] . Researchers have not yet discovered ongoing transmission of MERS-CoV within communities outside of health care settings.
The clinical presentation of MERS varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ARDS), septic shock, and multiple organ failure, often resulting in death. Most patients with MERS develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . Progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . MERS progresses much more rapidly to respiratory failure and has a higher case fatality rate than SARS [1] . Unlike SARS, however, infection with MERS-CoV is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . The overall CFR of MERS is approximately 34.4% [46] . Most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. As a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. Pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of MERS-CoV infection; however, little is known of the pathophysiology of this infection during pregnancy.
Limited data exists on the prevalence and clinical features of MERS during pregnancy, birth, and the postnatal period. It is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the MERS-CoV and the severity of clinical illness [60] . Pregnant women infected with SARS-CoV, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that MERS-CoV could also lead to severe clinical outcomes in pregnancy. To date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes.
Between November 2012 and February 2016, there were 1308 cases of MERS reported by the Saudi Arabia Ministry of Health (MoH). Of these, 5 patients were pregnant, according to a retrospective study by Assiri et al. [47] , and all resulted in adverse outcomes. Patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. All 5 cases received intensive care. Two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. These instances of severe maternal and perinatal outcomes are consistent with other reports of MERS-CoV infection in pregnant women, as well as outcomes associated with SARS-CoV infection. The authors of the retrospectives study concede that unreported cases of MERS in pregnancy are likely due to lack of routine pregnancy testing [47] . They conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for MERS-CoV, to contribute to overall understanding of pathogenesis and epidemiological risk. Additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to MERS-CoV in healthcare settings.
In a separate case report of MERS occurring in pregnancy, Alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. In the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. Following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (ICU) and later recovered. The preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. In contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of MERS-CoV exposure, her young age, the use of steroids, and differences in immune response.
Alfaraj et al. [61] described 2 cases of maternal infection with MERS-CoV at the Prince Mohammed Bin Abdulaziz Hospital (PMAH) in Saudi Arabia. Maternal infection in both cases was confirmed by nasopharyngeal swab testing by RT-PCR. One patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. The second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. This woman presented to the hospital after contact with a MERS-CoV-infected person during an active outbreak. Both patients later tested negative for MERS-CoV and were subsequently discharged. The younger patient delivered a healthy, full-term infant. The status of the other delivery is unknown. Neither fetus was tested for MERS-CoV.
According to Payne et al. [62] , epidemiologic investigation of the 2012 MERS outbreak in Zarqa, Jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to MERS-CoV. The mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. On the 7th day of symptoms, she had a fetal death. The mother was confirmed to have antibody to MERS-CoV, and she self-reported having had unprotected contact with family members who later tested positive for the virus. This was the first documented occurrence of stillbirth during maternal infection with MERS-CoV.
On 24 November 2013, a 32-year-old pregnant woman in the United Arab Emirates (UAE) developed ARDS following admission to the ICU after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . Later that day, her baby was delivered by caesarean section and subsequent Apgar scores were within healthy range. The next day, RT-PCR evaluation revealed that the mother was positive for MERS-CoV. Despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. While the outcome for this mother was fatal, Malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . More research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. While few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] .
Outside of the Middle East the only confirmed case of MERS in pregnancy occurred in 2015 in South Korea. Jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having MERS. Despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. Subsequent testing of the infant's blood did not detect any IgG, IgM, or IgA antibodies to MERS-CoV.
The mean maternal age of the 11 confirmed maternal SARS cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. The source of infection in 2 of the cases was attributed to contact with family members who tested positive for MERS-CoV, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). Six patients required intensive care and 3 died. Of those who died, 2 were exposed to MERS-CoV in the 3rd trimester, and 1 was exposed during the 2nd trimester. The infant death rate for all 11 cases was 27%. Fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. According to Alfaraj et al. [61] , the CFR for the 11 infected women-also 27%-was not statistically different from the overall CFR of MERS in the general population (35%) (P = 0.75). Only 1 case resulted in both maternal and fetal death.
Similar to SARS in pregnancy, more research is needed to understand the pathogenesis and epidemiology of MERS in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. Few studies documented the presence of MERS-CoV antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. As such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] .
MERS prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . Since 2013, the Saudi Arabia MoH has recommended that pregnant women postpone travel to Saudi Arabia for the Hajj and Umrah [47] . To further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. Pregnant women who present with symptoms of pneumonia, influenza-like illness (ILI), or sepsis on the Arabian Peninsula may also benefit from MERS-CoV screening to expedite early diagnosis and improve disease management [60] .
While multiple agents have been used to treat MERS, none have been tested in large clinical studies. Available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . A prospective or randomized study may prove difficult given the sporadic nature of MERS-CoV outbreaks.
Due to a gap in research on the treatment of MERS in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . Therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. For example, during the 2003 SARS outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] .
The Alphacoronaviruses HCoV 229E and NL63, as well as the Betacoronaviruses HKU 1 and OC43, can infect humans and cause the common cold. In order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, Gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. These specimens were evaluated for the presence of HCoV 229E, OC-43, NL63 and HKU 1 using RT-PCR methodology. Between the period from July 2003 to August 2005 the authors examined 159 mother-infant dyads. Human coronaviruses were identified in 12 samples (HCoV 229E: 11; HKU 1 : 1) from 7 mother-child pairs. In 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. There were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus.
It is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. There are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to SARS-CoV, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] .
It is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-nCoV. In examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. Today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . Excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] .
On 23 January 2020 the Coalition for Epidemic Preparedness Innovations (CEPI) announced three programs to develop a vaccine against the novel Wuhan coronavirus. The Chief Executive Officer of CEPI, Richard Hatchett, said [72] :
"Given the rapid global spread of the nCoV-2019 virus the world needs to act quickly and in unity to tackle this disease. Our intention with this work is to leverage our work on the MERS coronavirus and rapid response platforms to speed up vaccine development."
The novel coronavirus is the first epidemic disease to emerge since the formation of CEPI in Davos in 2017. CEPI was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. In May 2017, WHO released the Target Product Profile (TPP) for MERS-CoV vaccines, following the prioritization of MERS-CoV as one of eight priority pathogens for prevention of epidemics [73] . CEPI and partners aim to use existing platforms-that is, the existing "backbone" that can be adapted for use against new pathogens-that are currently in preclinical development for MERS-CoV vaccine candidates. Following the WHO declaration on 30 January that the current 2019-nCoV outbreak is a public health emergency of international concern (PHEIC), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease.
A critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? As of the PHEIC declaration, 12 groups have announced that they are developing new vaccines against 2019-nCoV and seven others announced initiatives to develop new therapies [74] . Safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. To date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rVSV-ZEBOV vaccine through 3 Ebola virus epidemics serves as a recent example [69] [70] [71] . Given the potential severity in pregnancy, as demonstrated by this review of maternal infections of SARS and MERS, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses.
On 5 February 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in Wuhan had tested positive for 2019-nCoV at the Wuhan Children's Hospital in Hubei Province 30 hours following its birth. According to the official Xinhua news agency, the infant was delivered on 2 February to a mother who had tested positive for the virus. Reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . Dr. Zeng Lingkong, Chief Physician at the Neonatal Medicine Department of the hospital, said [78] , "This reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission"
The hospital also provided information about a previous case of a baby that had been delivered on 13 January 2020. Following its birth, the infant's nanny was diagnosed with 2019-nCoV, and the mother was diagnosed days later [76] . On 29 January the baby began to develop symptoms. According to Dr. Zeng Lingkong [76] , "Whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. But we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected"
In considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. In some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. Neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. Based upon past experience with pregnant women who developed MERS and SARS, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. Discussing the most recent baby to be diagnosed with the 2019-nCoV infection, Dr. Stephen Morse, an epidemiologist at the Mailman School of Public Health at Columbia University stated [77] , "It's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat," "It's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing."
And according to Dr. Paul Hunter, Professor of Medicine at the University of East Anglia [79] , "As far as I am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. When a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth."
There is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, SARS and MERS. These previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. Both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. Coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the ICU, spontaneous abortion and perinatal death. Unlike some viral infections, notably Ebola virus [70] and Zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either SARS or MERS. It remains to be seen during the current Wuhan 2019-nCoV epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. However, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-nCoV. Additional clinical research on the treatment of SARS, MERS, and the new coronavirus 2019-nCoV is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. This research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks. | 2,551 | What animals can carry coronavirus? | {
"answer_start": [
2039
],
"text": [
"uding birds, farm animals, pets, camels, an"
]
} | 2,200 |
891 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the mean duration of time from single lobe consolidation to bilateral multilobar lung infiltrates in human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
1760
],
"text": [
"2 days"
]
} | 3,240 |
892 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the mean duration of time from first positive chest x-ray to bilateral multilobar lung infiltrates in human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
1771
],
"text": [
"4.8 days"
]
} | 3,241 |
893 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What are the most frequent clinical manifestations of human adenovirus type 55 (HAdV-55) induced ARDS? | {
"answer_start": [
2285
],
"text": [
"Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates"
]
} | 3,242 |
894 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What do we know about the genomics of human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
3601
],
"text": [
"This pathogen was fully characterized by whole-genome sequencing"
]
} | 3,243 |
895 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What are the clinical symptoms of human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
8509
],
"text": [
"Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness"
]
} | 3,244 |
896 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the mean time from onset of symptoms to dyspnea in human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
8958
],
"text": [
"5 days"
]
} | 3,245 |
897 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the mean time of onset of symptoms to ICU admission in human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
9127
],
"text": [
"9.6 days"
]
} | 3,246 |
898 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the mean rate of respiration upon admission to the ICU when admitted for human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
9244
],
"text": [
"43 breaths per minute"
]
} | 3,247 |
899 | Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/
SHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0
Authors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen
Date: 2014-08-12
DOI: 10.1186/s13054-014-0456-6
License: cc-by
Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was 5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012
Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .
Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.
Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.
Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.
Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.
Viral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.
Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).
During the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.
All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.
Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.
Four patients had lower than normal T-cell subset counts (Table 2) .
CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).
All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).
Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.
All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.
To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support.
Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.
The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.
Our study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .
Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the reaction to therapy and patient outcome.
The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.
Our study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response. | 1,604 | What is the white blood cell count in severe cases of human adenovirus type 55 (HAdV-55)? | {
"answer_start": [
9441
],
"text": [
" low or in the normal range"
]
} | 3,248 |
Subsets and Splits