The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.

Viruses are ubiquitous biological entities whose importance and role in aquatic habits is beginning to take form. However, several habitats have undergone limited to no examination with viral-geochemical parameters minimally examined and viral-mineral relationships in the natural environment and the role of mineralization on viral-host dynamic completely lacking. To further develop knowledge on the presence and abundances of viruses, how viruses impact aquatic systems, and how viral-host interactions can be impacted under mineralizing conditions, viruses were examined under a variety of habitats and experimental conditions. Water samples were collected from the deep subsurface (up to 450 m underground) and acid mine drainage (AMD) systems in order to determine the presence, abundance, and viral-geochemical relationships within the systems. Samples were also collected from a variety of freshwater habitats, which have undergone limited examination, to determine viral-geochemical and viral-mineral relationships. Lastly, bacteriophage-host dynamics were examined under authigenic mineral precipitation to determine how mineralization impacts this relationship. Results reveal that not only are viruses present in the deep subsurface and AMD systems, but they are abundant (up to 107 virus-like particles/mL) and morphogically diverse. Viruses are also the strongest predictor of prokaryotic abundance in southern Ontario freshwater systems where potential nutrients are rich. Geochemical variables, such as pH and Eh, were shown to have negative impacts of viral abundance indicting that AMD environments are detrimental for free viruses (i.e. not particle associated). Direct evidence of viral-mineral interactions was found using transmission electron microscopy as viral particles were shown attached to iron-bearing mineral phases (determined through elemental analysis). In addition, evidence of viral participation in mineralization events was found in both AMD and freshwater environments where inverse correlations were noted between viral abundance and jarosite saturation indices (r = -0.71 and r = -0.33, respectively), and goethite saturation indices were also noted to be the strongest predictor of VLP abundance in freshwater habitats explaining 78% of the variability in the data. Lastly, iron precipitation and/or metal ion binding to bacterial surfaces greatly reduced phage replication (~98%) revealing bacterial mineralization has a protective benefit strongly hindering viral replication.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Influenza is a contagious respiratory disease that has caused at least four pandemics and countless epidemics since the 20th century, impacted millions of people worldwide and the global economy. To date, the predominant influenza species circulating in humans are influenza A and B. Influenza may cause serious illness in all age groups but individuals such as the newborns and senior population whose immune systems are compromised are at higher risk for severe disease. Interestingly, during the outbreak of pandemic 2009 H1N1 (H1N1pdm), it was found that the elderly had the lowest hospitalization rate and an increased proportion of healthy adults developed severe disease. Furthermore, several clinical studies have demonstrated that most H1N1pdm infected children experienced mild to moderate illness and led to the least mortality. The difference of disease outcome in age groups between different influenza infections may be due to several factors, which include differing pathogenicity between the viruses, differential immune status and composition among the age groups, and pre-existing immunity from previous encounter(s) with a similar virus. Since the human clinical data are often complicated by secondary factors such as co-morbidities, I used the ferret model to address these questions. I first compared the clinical and pathological patterns among the pandemic and seasonal influenza strains and found H1N1pdm caused the most severe illness to healthy ferrets. Importantly, the disease severity did not correlate with viral burden but immunopathology. To study the age effect, I found that H1N1pdm infected young ferrets with mild clinical symptoms developed specialized ectopic lymphoid structures and a distinct cytokine expression profile in the lungs, which were absent in adult ferrets with severe illness. I also examined antigenic change in historical H1N1s and anti-H1 responses to explain the pre-existing immunity of H1N1pdm found in the elderly. However, low similarity was found between historical H1N1s and H1N1pdm. Lastly, I conducted a detailed influenza B comparative study. I observed the pathogenic B strain was capable to cause lower respiratory tract infection and pathology like the influenza A viruses. Overall, this thesis provides novel insights for developing therapeutic and prophylactic strategies against influenza infection.

Réovirus, connu sous le nom REOLYSIN®, est présentement à l'étude à titre d'agent oncolytique. Or, la spécificité du virus pour les cellules cancéreuses pourrait être optimisée par une modification au niveau de la protéine d'attachement σ1. La présente étude vise à démontrer qu'une telle amélioration est possible par l'utilisation de la méthode nouvellement décrite de génétique inverse. Par cette technique, il est possible d'ajouter un polypeptide d'une longueur de quarante acides aminés à l'extrémité C-terminale de σ1. Il est aussi possible d'engendrer des virus mutés en leur site d'activité mucinolytique. Les virus nouvellement créés démontrent une efficacité de réplication diminuée, mais demeurent infectieux. Contrairement aux méthodes traditionnellement utilisées avec réovirus, la méthode de génétique inverse permet de conserver les mutations engendrées, par substitution ou addition, au cours des cycles de réplication. Une telle étude démontre qu'il serait possible de modifier le tropisme de réovirus. / Reovirus, also known under the tradename REOLYSIN®, is currently under clinical trial as an oncolytic agent. The specificity of the virus for transformed (cancerous) cells could be improved by modifications of the σ1 attachment protein. This study presents two ways to achieve this improvement by using a newly developed method of reverse genetics for double-stranded RNA viruses. As a proof of concept, we engineered the addition of an exogenous polypeptide of forty amino-acids to the C-terminal end of σ1. Subsequently, we created viruses with compromised mucinolytic activities by introducing amino-acids substitutions in the appropriate genetic sites. A decreased replicative efficacy was observed in all experimentally generated viruses, despite maintaining their infectious capacity. Reverse genetics allows the generation of viruses that retain their mutations throughout successive replicative cycles, which was impossible with traditionnal techniques. This project demonstrates the feasibility of tropism modification by using the reverse genetics method.

GB virus C (GBV-C) is a common, apathogenic virus that can inhibit human immunodeficiency virus (HIV) replication in vitro. Persistent coinfection with GBV-C has been associated with improved survival among HIV-infected adults while loss of GBV-C viremia has been associated with poor survival. If GBV-C does inhibit HIV replication, it is possible that GBV-C infection may reduce mother-to-child-transmission (MTCT) of HIV. This study investigated whether maternal or infant GBV-C infection was associated with reduced MTCT of HIV infection. The study population consisted of 1,783 pregnant women from three Bangkok perinatal HIV transmission studies (1992-94, 1996-7, 1999-2004). We tested plasma collected at delivery for GBV-C RNA, GBV-C antibody, and GBV-C viral genotype. If maternal GBV-C RNA was detected, the four- or six-month infant specimen was tested for GBV-C RNA. Rates of MTCT of HIV in GBV-C-infected and GBV-C-uninfected women and infants were compared using multiple logistic regression as were associations with MTCT of GBV-C and prevalence of GBV-C infection. The prevalence of GBV-C infection (i.e. presence of RNA or antibody) was 33% among HIV-infected women and 15% among HIV-uninfected women. Forty-one percent of GBV-C-RNA-positive women transmitted GBV-C to their infants. Only two of 101 (2.0%) GBV-C-RNA-positive infants acquired HIV infection compared to 162 (13.2%) of 1,232 of GBV-C-RNA-negative infants (RR 0.15, p<0.0001). This association remained after adjustment for maternal HIV viral load, antiretroviral prophylaxis, CD4+ count and other covariates. MTCT of HIV was not associated with presence of maternal GBV-C RNA or maternal GBV-C antibody. Maternal receipt of antiretroviral therapy was associated with increased MTCT of GBV-C, as was high GBV-C viral load, vaginal delivery and absence of infant HIV infection. GBV-C infection among women was independently associated with more than one lifetime sexual partner, intravenous drug use and HIV-infection. We observed a higher prevalence of GBV-C infection among HIV-infected compared to HIV-uninfected pregnant women in Thailand, likely due to common risk factors. Antiretroviral therapy appears to increase MTCT of GBV-C. Infant GBV-C acquisition, but not maternal GBV-C infection, was significantly associated with reduced MTCT of HIV. Mechanisms for these later two associations are unknown. / Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero J, Kaul R, Kulkarni P, McConnell MS, Mock PA, Culnane M, McNicholl J, Roongpisuthipong A, Chotpitayasunondh T, Shaffer N, Butera S. 2008. Reduced mother-to-child transmission of HIV associated with infant but not maternal GB virus C infection. J Infect Dis 197(10):1369-1377. Bhanich Supapol W, Remis RS, Raboud J, Millson M, Tappero JW, Kaul R, Kulkarni P, McConnell MS, Mock PA, McNicholl JM, Vanprarar N, Asavapiriayanont S, Shaffer N, Butera ST. 2009. Mother-to-child transmission of GB virus C in a cohort of women coinfected with GB virus C and HIV in Bangkok, Thailand. J Infect Dis 200:227-235.

L’infection par le Virus Respiratoire Syncytial cause des affections pulmonaires aiguës en pédiatrie caractérisée par une réponse inflammatoire excessive médiée par la production de cytokines par les cellules épithéliales des voies aériennes. Les gènes codant pour ces cytokines sont régulés par le facteur de transcription NF-κB (p50/p65) dont l’activation est classiquement induite par la phosphorylation de son inhibiteur IκBα, ce qui permet l’accumulation de l’hétérodimère au noyau. Par contre, nous avons récemment identifié la phosphorylation en sérine 536 de la sous-unité p65 comme une autre étape essentielle à son activation lors de l’infection des AEC par RSV. Le travail présenté dans ce mémoire a permis de démontrer que l’inhibition de l’expression de RIG-I, de Cardif ou de TRAF6, 3 protéines impliquées dans la reconnaissance cellulaire des virus, conduit à l’inhibition de cette phosphorylation en réponse à RSV. Nous avons également établi à l’aide d’inhibiteurs pharmacologiques et d’ARNi que, parmi les diverses kinases connues pour phosphoryler p65 en réponse à divers stimulus, IKKα/β sont essentielles à cette phosphorylation lors d’une stimulation par RSV. Puisque TRAF6 est bien connu dans la littérature pour activer le complexe IKK, nous proposons que TRAF6, après reconnaissance de l’ARN viral de RSV par RIG-I, active le complexe IKK qui induit la phosphorylation de la sousunité p65 de NF-κB, permettant l’expression de gènes cibles. D’autre part, nous avions précédemment démontré que Nox2, un isoforme de NADPH oxydase, contrôle l’activation de NF-κB en régulant les phosphorylations de IκBα et p65. Nous montrons ici que l’inhibition de Nox2 réduit fortement l’activité du complexe kinase IKK. De plus, la présence au niveau basal de Nox2 est critique pour le niveau d’ARN messager de Cardif. Nous proposons donc que la régulation de la phosphorylation de p65 en ser536 par Nox2 soit via son effet sur Cardif en permettant la fonctionnalité de la voie RIG-I. / The infection by the Respiratory Syncytial Virus causes acute respiratory tract affections among children characterized by an excessive inflammatory response mediated by airway epithelial cells production of cytokines. The genes coding for theses cytokines are regulated by the transcription factor NF-κB (p50/p65) which is classically activated by phosphorylation of its inhibitor IκBα, permitting the nuclear accumulation of the heterodimer. The work presented in this master’s thesis allowed demonstrating that the inhibition of either RIG-I, Cardif or TRAF6, 3 proteins implicated in the cellular recognition of virus, leads to the inhibition of this phosphorylation in response to RSV. Moreover, we established with pharmacological inhibitors and siRNA that, among all kinases known to phosphorylate p65 in response to various stimulus, IKKα/β are essential to this phosphorylation in RSV stimulation. Since TRAF6 is a well-known IKK complex activator in the literature, we propose that TRAF6, after the recognition of the RSV viral RNA by RIG-I, activates the IKK complex witch induces the phosphorylation of the NF-κB subunit p65, allowing the expression of targets genes. Furthermore, we had previously demonstrated that Nox2, a NADPH oxydase isoform, controls the activation of NF-κB by regulating the phosphorylation of IκBα and p65. We show here that the inhibition of Nox2 by siRNA reduces strongly the activity of the IKK complex. Moreover, the basal level presence of Nox2 is critic for the messenger RNA level of Cardif. Thus, we propose that the Nox2 regulation of p65 ser536 is done by its effect on Cardif, which allows the integrity of the RIG-I pathway.

La prolifération, la différenciation ainsi que les fonctions des cellules du système immunitaire sont contrôlées en partie par les cytokines. Lors de l’infection par le VIH-1, les défauts observés dans les fonctions, la maintenance, ainsi que la consistance des cellules du système immunitaire sont en large partie attribués à une production altérée des cytokines et à un manque d’efficacité au niveau de leurs effets biologiques. Durant ces études, nous nous sommes intéréssés à la régulation et aux fonctions de deux cytokines qui sont l’IL-18 et l’IL-21. Nous avons observé une corrélation inversée significative entre les concentrations sériques d’IL-18 et le nombre des cellules NK chez les patients infectés par le VIH-1. Nos expériences in vitro ont démontré que cette cytokine induit l’apoptose des cellules NK primaires et que cette mort peut être inhibée par des anticorps neutralisants spécifiques pour FasL et TNF-α. Cette mort cellulaire est due à l’expression de FasL sur les cellules NK et à la production de TNF-α par ces cellules. L’IL-18 augmente aussi la susceptibilité à la mort des cellules NK par un stimulus pro-apoptotique, en diminuant l’expression de la protéine anti-apoptotique Bcl-XL. Nous démontrons aussi que, contrairement à l’IL-18, les niveaux d’IL-18BP sont plus faibles dans les sérum de patients infectés. Ceci résulte sur une production non coordonnée de ces deux facteurs, aboutissant à des niveaux élevés d’IL-18 libre et biologiquement active chez les patients infectés. L’infection de macrophages in vitro induit la production d’IL-18 et réduit celle d’IL-18BP. De plus, l’IL-10 et le TGF-β, dont les concentrations sont élevées chez les patients infectés, réduisent la production d’IL-18BP par les macrophages in vitro. Finalement, nous démontrons que l’IL-18 augmente la réplication du VIH-1 dans les lymphocytes T CD4+ infectés. Les niveaux élevés d’IL-18 libres et biologiquement actives chez les patients infectés contribuent donc à l’immuno-pathogénèse induite par le VIH-1 en perturbant l’homéostasie des cellules NK ainsi qu’en augmentant la réplication du virus chez les patients. Ces études suggèrent donc la neutralisation des effets néfastes de l’IL-18 en utilisant son inhibiteur naturel soit de l’IL-18BP exogène. Ceci permettrait de moduler l’activité de l’IL-18 in vivo à des niveaux souhaitables. L’IL-21 joue un rôle clef dans le contrôle des infections virales chroniques. Lors de ces études, nous avons déterminé la dynamique de la production d’IL-21 lors de l’infection par le VIH-1 et sa conséquence sur la survie des cellules T CD4+ et la fréquence des cellules T CD8+ spécifiques au VIH-1. Nous avons démontré que sa production est compromise tôt au cours de l’infection et que les concentrations d’IL-21 corrèlent avec le compte de cellules T CD4+ chez les personnes infectées. Nos études ont démontré que le traitement antirétroviral restaure partiellement la production d’IL-21. De plus, l’infection par le VIH-1 de cellules T CD4+ humaines inhibe sa production en réduisant l’expression du facteur de transcription c-Maf. Nous avons aussi démontré que la fréquence des cellules T CD4+ spécifiques au VIH-1 qui produisent de l’IL-21 est réduite chez les patients virémiques. Selon nos résultats, l’IL-21 empêche l’apoptose spontanée des cellules T CD4+ de patients infectés et l’absence d’IL-21 réduit la fréquence des cellules T CD8+ spécifiques au VIH-1 chez ces patients. Nos résultats démontrent que l'IL-21R est exprimé de façon égale sur tous les sous-types de cellules NK chez les donneurs sains et chez les patients infectés. L’IL-21 active les protéines STAT-3, MAPK et Akt afin d'augmenter les fonctions effectrices des cellules NK. L'activation de STAT-3 joue un rôle clef dans ces fonctions avec ou sans un traitement avec de l'IL-21. L'IL-21 augmente l'expression des protéines anti-apoptotiques Bcl-2 et Bcl-XL, augmente la viabilité des cellules NK, mais ne possède aucun effet sur leur prolifération. Nous démontrons de plus que l'IL-21 augmente l'ADCC, les fonctions sécrétrices et cytotoxiques ainsi que la viabilité des cellules NK provenant de patients chroniquement infectés par le VIH-1. De plus, cette cytokine semble présenter ces effets sans augmenter en contrepartie la réplication du VIH-1. Elle permet donc d'inhiber la réplication virale lors de co-cultures autologues de cellules NK avec des cellules T CD4+ infectées d'une manière dépendante à l'expression de perforine et à l'utilisation de la protéine LFA-1. Les niveaux d’IL-21 pourraient donc servir de marqueurs biologiques pour accompagner les informations sur le taux de cellules T CD4+ circulantes en nous donnant des informations sur l’état de fonctionnalité de ce compartiment cellulaire. De plus, ces résultats suggèrent l’utilisation de cette cytokine en tant qu’agent immunothérapeutique pour restaurer les niveaux normaux d’IL-21 et augmenter la réponse antivirale chez les patients infectés par le VIH-1. / The proliferation, differentiation, function and maintenance of immune cells is controlled in large part by cytokines. HIV-induced dysfunctions of the antiviral immunity is in part related to defects in the cytokine network, as manifested by altered cytokine secretion and responsiveness to these cytokines. In these studies, we investigated the regulation and the functions of two cytokines, IL-18 and IL-21, during HIV-1 infection. In our studies, we observed an inverse correlation between IL-18 concentrations and absolute numbers of various subsets of NK cells in infected persons. IL-18 caused increased death of a human NK cell line, as well as of primary human NK cells in vitro. The IL-18-mediated cell death was dependent upon Fas-FasL interactions and TNF- secretion. IL-18 induced the expression of TNF-, induced the expression of FasL on NK cells, increased the transcription from the human FasL promoter, reduced the expression of Bcl-XL in NK cells, and increased their sensitivity to FasL-mediated cell death. In contrast to IL-18 levels, IL-18BP levels decreased in the serum of HIV-infected patients. This decrease resulted in enhanced levels of free IL-18 in the serum of such patients. The infection increased production of IL-18 but decreased that of IL-18BP in monocyte-derived macrophages (MDM). Furthermore, IL-10 and TGF-β, two cytokines for which concentrations are increased in HIV-infected persons, also decreased production of IL-18BP by human MDM. Finally, recombinant human IL-18 enhanced HIV-1 replication in human CD4+ T cells. The uncoordinated production of these two cytokines represents an imbalance between these two soluble factors in HIV-infected patients. Our study shows that enhanced IL-18 bioactivity in HIV-infected patients may contribute to the pathogenesis of AIDS by disrupting NK cell homoeostasis and increasing viral replication. This uncoordinated production of IL-18 and IL-18BP contribute to IL-18-induced immunopathology and pathogenesis in HIV-infected AIDS patients. Therefore, these studies suggest that the neutralization of IL-18 may represent an appropriate and useful immunotherapeutic strategy in these patients. It may delay AIDS progression and improve the immune status of infected persons. The best way to achieve this goal may be using exogenous interleukin-18 binding protein. IL-21 is a relatively newly discovered immune enhancing cytokine, which plays an essential role in controlling chronic viral infections. Therefore, we sought to determine the dynamics of the cytokine production and its potential consequences on the viability of CD4+ T cells in HIV-infected persons. We show here that the cytokine production is compromised early in the course of the infection. The serum cytokine concentrations correlated with CD4+ T cell counts in the infected persons. Among different groups of HIV-infected persons, only Elite Controllers maintain normal production of the cytokine. The HAART partially restores production of this cytokine. Interestingly, HIV-1 infection of human PBMC as well as of purified CD4+ T cells inhibits the production of the cytokine by decreasing the expression of c-Maf, a transcription factor involved in the activation of the cytokine gene, in the virus-infected cells but not in uninfected bystander cells. We also show that the frequencies of IL-21 producing HIV-specific antigen experienced CD4+ T cells are decreased in HIV-infected viremic patients. Furthermore, we show that recombinant human IL-21 acts as pro-survival factor and prevents enhanced spontaneous apoptosis of ex vivo cultured CD4+ T cells from HIV-infected patients and that increased serum levels of the cytokine are associated with higher frequencies as well as with better functions of HIV-specific CTL in HIV-infected individuals. We show that the cytokine receptors are expressed equally on all NK cell subsets. We demonstrate that the cytokine activates STAT-3, MAPK and Akt to enhance NK cell functions. IL-21 increases expression of anti-apoptotic proteins Bcl-2 and Bcl-XL, and enhances viability of NK cells, but has no effect on their proliferation. We further show that the cytokine enhances HIV-specific ADCC, secretory and cytotoxic functions as well as viability of NK cells from HIV-infected persons. Furthermore, it exerts its biological effects on NK cells with minimal enhancement of HIV-1 replication, and the cytokine-activated NK cells inhibit viral replication in co-cultured HIV-infected autologous CD4+ T cells in a perforin- and LFA-1-dependent manner. These studies suggest that serum IL-21 concentrations may serve as useful biomarker to accompany CD4+ T cell counts for monitoring HIV-1 disease progression and the fitness of the antiviral immunity. Furthermore, the cytokine may be considered for immunotherapy in HIV-infected patients in order to restore the physiological levels of the cytokine and promote their antiviral immunity.