Alagarsamy Srinivasan

Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr

Vpr, a 14-kDa, 96 amino acid protein, is conserved among the primate lentiviruses HIV-1, HIV-2 and Simian Immunodeficiency virus supporting the notion that it plays an important role in virus life cycle in vivo. Vpr appears to have several functions including cell cycle arrest at G2 stage, apoptosis, nuclear localization, nuclear import of the pre-integration complex, cation selective channel activity and transcriptionally activate HIV-1 LTR and other heterologous promoters. Over the years, we have addressed several issues pertaining to Vpr including the amount of Vpr present in the virus particles and structure-function relationship of Vpr. Here, we have reviewed the sources of Vpr that may potentially contribute to the cytopathic features observed in the context of HIV-1 infection. There are three different sources of Vpr available in the infected individuals to initiate the pathogenic effects. These include cell-associated, virion-associated (infectious, infectious-non productive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). A potential role of Vpr in neuropathogenesis of HIV infection in CNS was also suggested by early studies demonstrating neurotoxicity of recombinant Vpr protein. Interestingly, free Vpr (cell-free and virus-free) has been demonstrated in the serum of HIV-1 infected individuals and in the CSF of AIDS patients with neurological dysfunctions. Based on the toxic effects of extra-cellular Vpr on cells noted in several studies, it is likely that free Vpr could contribute to the bystander cell depletion in lymphoid tissues, peripheral blood, and the CNS. These results led us to propose a model for the role of Vpr in AIDS pathogenesis.

Transdominant Activity of Human Immunodeficiency Virus Type 1 Vpr with a Mutation at Residue R73

The 96-amino-acid-long human immunodeficiency virus type 1 virion-encoded accessory protein Vpr is of particular interest, as this protein, which is found in association with viral particles, can exert a regulatory effect on both virus replication and host cell function. Evidently, Vpr, through interaction with several host regulatory proteins, can modulate transcription from the viral long terminal repeat promoter. Expression of Vpr in cells results in deregulation of cell proliferation during the cell cycle pathway at the G(2) stage. Vpr has unique structural features consisting of multiple functional domains. In this study, we have focused on the leucine/isoleucine-rich domain near the carboxyl terminus of Vpr at residue 73 (arginine) and have demonstrated that alterations at this residue result in ablation of transcriptional activity of Vpr and its ability to block cell cycle events at the G(2) stage. Interestingly, substitution mutations at R73 have resulted in a peptide with dominant negative activities on wild-type functions in transcription and host proliferation events. Results from in vitro and in vivo protein-protein interaction studies have revealed that functionally inactive mutant Vpr can be associated with wild-type protein, presumably through the N-terminal regions of the protein which have been shown to be important for Vpr oligomerization. Thus, it is likely that complexation of the mutant Vpr with wild-type protein functionally inactivates Vpr. The importance of these findings in light of the development of therapeutic strategies is discussed.

Functional Role of Residues Corresponding to Helical Domain II (Amino Acids 35 to 46) of Human Immunodeficiency Virus Type 1 Vpr

ABSTRACT Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G2, nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLΔVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprΔ35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike other mutants and control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G2, mutant Vpr showed an intermediary effect with the exception of VprΔ35-50 and VprΔ35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.

Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells

BackgroundHuman immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages.MethodsMonocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines.ResultsHIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors.ConclusionCollectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.

HIV-1 Mediated Immune Pathogenesis: Spotlight on the Role of Viral Protein R (VPR)

HIV-1 exploits the cellular machinery to replicate in the host cells by targeting a wide range of host factors at different stages of its life cycle. HIV-1 also induces detrimental effects in the infected and uninfected bystander cells resulting in dysregulation including interference in immune effector functions. The latter is specifically linked to the immune evasion strategies of the virus. In addition to the essential roles of structural proteins (Gag, Pol & Env), HIV-1 encoded auxiliary proteins such as Nef, Vif, Vpu, and Vpr through their interaction with the host cellular partners facilitate viral replication and dissemination. HIV-1 Vpr, a virion-associated molecule, has been implicated to play a role in the early events in virus life cycle. Vpr is a pleiotropic protein that exerts a range of effects including inhibition of cell proliferation, induction of apoptosis and modulation of a number of immune molecules. These functions could be in part responsible for Vpr induced immune evasion and virus replication. Appreciating this view is the genetic variation in vpr gene reflected in the form of polymorphisms at the amino acid level that may contribute to the potential CTL escape of the virus. It is likely that Vpr mediated dysregulation of host immune response contributes, in part, to the progression of disease. This review focuses on the recent advances regarding HIV-1 Vpr mediated immunopathogenesis and the mechanistic insight from in vitro and in vivo studies.

Human immunodeficiency virus type 1 Vpr: oligomerization is an essential feature for its incorporation into virus particles

HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to its functions are not clear. To address this, we have utilized molecular modeling approaches to identify putative models of oligomerization. The predicted interface residues were subjected to site-directed mutagenesis and evaluated their role in intermolecular interaction and virion incorporation. The interaction between Vpr molecules was monitored by Bimolecular Fluorescence complementation (BiFC) method. The results show that Vpr forms oligomers in live cells and residues in helical domains play critical roles in oligomerization. Interestingly, Vpr molecules defective in oligomerization also fail to incorporate into the virus particles. Based on the data, we suggest that oligomerization of Vpr is essential for virion incorporation property and may also have a role in the events associated with virus infection.

The role of Vpr in the regulation of HIV-1 gene expression.

Expression of the viral protein R, Vpr, of HIV-1 affects many biological events in host cells including cell cycle progression, and modulates HIV-1 gene transcription. Earlier studies implicated the cellular protein p21WAF1 (p21) in regulating HIV-1 transcription, which led us to investigate the functional and physical interaction of Vpr and p21. Our results show that Vpr modestly activated HIV-LTR in cells lacking p21 gene. We described the mechanisms of p21 and Vpr interaction for stimulating transcription of HIV-1. Data from the protein-protein interaction experiments revealed the ability of Vpr, p21 and p300 to form a complex. Further, we showed that, Vpr interacted with the N- and the C-terminal domains of p21. Furthermore, in cells expressing Vpr, p21 localizes to both the cytoplasm and the nucleus. Additionally, expression of Vpr alleviates p21-mediated inhibition of cell departure from G1 phase. Expression of a mutant Vpr, with arginine 73 altered to serine, did not affect p21 ability to cause cell arrest or its sub- cellular localization. These observations revealed a new cellular partner for Vpr, and provided a new therapeutic avenue for controlling HIV-1 expression.

HIV-1 Vpr: a closer look at the multifunctional protein from the structural perspective.

The human immunodeficiency virus-1 (HIV-1) Vpr protein plays multiple roles in HIV-1 replication. In early infection, Vpr provides help in the nuclear localization of pre-integration complex. Subsequently, Vpr induces cell cycle arrest of infected cells at G2/M phase. Cell cycle arrest facilitates higher rate of viral gene transcription. Vpr is also capable of activating transcription of viral and heterologous genes. Vpr induces apoptosis in infected cells leading to loss of immune cells and onset of clinical AIDS. Interestingly, Vpr is also considered as a passenger protein in the virus particles as it is incorporated into the virus particles through interaction with Gag. The structure of full length Vpr has been resolved recently through NMR. In this review, we have analysed the functions of Vpr using the available data from structural perspective. Packing of the three helices of Vpr around a core formed by hydrophobic side chains and integrity of helical domains are critical for Vpr functions. The distinct functions of Vpr have been attributed to structural integrity of different domains. The unique distribution of acidic and basic residues in Vpr is an interesting feature. Two hydrophobic pockets on the structure of Vpr are proposed to be important targets for modulating Vpr functions. The inter-relationship between different functions of Vpr is discussed in the context of structure. Based on bioinformatics analysis, we propose new targets for modulating Vpr functions, which need to be validated experimentally.

Human herpesvirus-8 encoded Kaposin: subcellular localization using immunofluorescence and biochemical approaches.

Human herpesvirus-8 (HHV-8) has been causally linked to the development of Kaposis sarcoma (KS). DNA sequence analysis of the viral genome revealed a total of 81 open reading frames (ORF). Interestingly, only a small subset of these ORFs has been shown to be transcribed in cells latently infected with HHV-8 and in cells of the KS lesions. Among the genes active during latency, kaposin, is noted for its abundance and ability to transform cells in culture, thus implicating a potential role in KS pathogenesis. This has prompted us to undertake an investigation on elucidating the mechanism(s) by which Kaposin brings about transformation of cells. Towards this goal, we have generated an eukaryotic expression plasmid encoding Kaposin (Kap). As Kaposin is predicted to be a type II membrane protein, several strategies were utilized to address this, including the generation of Kaposin with the Flag (FL) epitope (DYKDDDDK) at the C-terminus of the protein (Kap-C-FL). Antibodies specific for Kaposin (kap-2), recognized both Kaposin and Kaposin-Flag, while antibodies against the Flag epitope recognized only Kaposin-Flag. Transfection of Kap and Kap-C-FL expression plasmid DNA into NIH3T3 cells resulted in cellular clones that exhibited a phenotypic property of transformation by forming large, multiclustered cells, when grown on soft agar. Because there is controversial data regarding the localization of Kaposin in cells, we examined the subcellular localization of Kaposin using confocal microscopy. We observed that Kaposin and Kaposin-Flag showed an intense staining surrounding the nucleus. Although there was no staining at the cell membrane of transfected cells, FACS analysis using kap-2 or Flag antibodies, under nonpermeable conditions, showed positivity. Cell fractionation studies further showed that the majority of Kaposin was detected in the nuclear fraction by Western blot analysis. The cytoplasmic and detergent soluble membrane fractions did not show Kaposin protein; however, a small amount was detected in the detergent insoluble membrane fraction. Taken together, these results suggest that Kaposin exhibits multicompartmental localization in cells.

Combined effects of hyperglycemic conditions and HIV-1 Nef: a potential model for induced HIV neuropathogenesis

Hyperglycemic conditions associated with diabetes mellitus (DM) or with the use of antiretroviral therapy may increase the risk of central nervous system (CNS) disorders in HIV-1 infected patients. In support of this hypothesis, we investigated the combined effects of hyperglycemic conditions and HIV-1 accessory protein Nef on the CNS using both in vitro and in vivo models. Astrocytes, the most abundant glial cell type required for normal synaptic transmission and other functions were selected for our in vitro study. The results show that in vitro hyperglycemic conditions enhance the expression of proinflammatory cytokines including caspase-3, complement factor 3 (C3), and the production of total nitrate and 8-iso-PGF2 α as reactive oxygen species (ROS) in human astrocytes leading to cell death in a dose-dependent manner. Delivery of purified recombinant HIV-1 Nef protein, or Nef expressed via HIV-1-based vectors in astrocytes showed similar results. The expression of Nef protein delivered via HIV-1 vectors in combination with hyperglycemia further augmented the production of ROS, C3, activation of caspase-3, modulation of filamentous protein (F-protein), depolarization of the mitochondria, and loss of astrocytes. To further verify the effects of hyperglycemia and HIV-1 Nef protein on CNS individually or in combination, in vivo studies were performed in streptozotocin (STZ) induced diabetic mice, by injecting HIV-1 Nef expressing viral particles into the sub-cortical region of the brain. Our in vivo results were similar to in vitro findings indicating an enhanced production of caspases-3, ROS (lipid oxidation and total nitrate), and C3 in the brain tissues of these animals. Interestingly, the delivery of HIV-1 Nef protein alone caused similar damage to CNS as augmented by hyperglycemia conditions. Taken together, the data suggests that HIV-1 infected individuals with hyperglycemia could potentially be at a higher risk of developing CNS related complications.