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Erwann Le Rouzic - One of the best experts on this subject based on the ideXlab platform.
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molecular insight into how hiv 1 Vpr Protein impairs cell growth through two genetically distinct pathways
Journal of Biological Chemistry, 2011Co-Authors: Erwann Le Rouzic, Claire Maudet, Matthieu Bertrand, Hichem LahouassaAbstract:Vpr, a small HIV auxiliary Protein, hijacks the CUL4 ubiquitin ligase through DCAF1 to inactivate an unknown cellular target, leading to cell cycle arrest at the G2 phase and cell death. Here we first sought to delineate the Vpr determinants involved in the binding to DCAF1 and to the target. On the one hand, the three α-helices of Vpr are necessary and sufficient for binding to DCAF1; on the other hand, nonlinear determinants in Vpr are required for binding to the target, as shown by using Protein chimeras. We also underscore that a SRIG motif conserved in the C-terminal tail of Vpr Proteins from HIV-1/SIVcpz and HIV-2/SIVsmm lineages is critical for G2 arrest. Our results suggest that this motif may be predictive of the ability of Vpr Proteins from other SIV lineages to mediate G2 arrest. We took advantage of the characterization of a subset of G2 arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G2 arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G2 arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G1 or S phases but trigger apoptosis through caspase 3. Disruption of DCAF1 binding restored efficiency of colony formation. However, DCAF1 binding per se is not sufficient to confer cytopathicity. These data support a model in which Vpr recruits DCAF1 to induce the degradation of two host Proteins independently required for proper cell growth.
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the human immunodeficiency virus type 2 vpx Protein usurps the cul4a ddb1dcaf1 ubiquitin ligase to overcome a postentry block in macrophage infection
Journal of Virology, 2009Co-Authors: Anna Bergamaschi, Erwann Le Rouzic, Marina Morel, Diana Ayinde, Annie David, Gilles Collin, Diane DescampsAbstract:Human immunodeficiency virus type 2 (HIV-2), the second causative agent of human AIDS (5), arose from cross-transmission of sooty mangabey simian immunodeficiency virus (SIVsm), a lentivirus that naturally infects sooty mangabeys without inducing overt disease (14). Although AIDS caused by HIV-2 is as lethal as that caused by HIV-1, most HIV-2 carriers remain asymptomatic much longer than HIV-1 carriers (36). On the other hand, cross-transmission of SIVsm to rhesus macaques gave rise to highly pathogenic viral strains, and infected animals develop a disease similar to human AIDS (14). Thus, the SIVsm lentiviral lineage exhibits markedly different pathogenicities in its original and recently acquired hosts. It is currently unknown whether cross-species transmission of primate lentiviruses leads to specific functional changes in viral Proteins that might directly modulate pathogenesis in the new host species. The HIV-1 and HIV-2 lentiviral lineages differ in the auxiliary Proteins encoded by their genomes, which likely reflects different selective pressures in their adaptation to the host cell environment. Thus, the Vpx Protein found in HIV-2 has no counterpart in HIV-1, whereas the genetically related Vpr Protein is present in both lineages (47). Both Vpx and Vpr are actively packaged into the virions (1, 40), which suggests a role in early infection steps, i.e., prior to viral synthesis. Lack of Vpx strongly decreases the pathogenicity of SIVsmPBj in infected macaques (17). In vitro, Vpx is dispensable for infection of immortalized T cells but has been shown to be critical for primary macrophage infection (9, 23). In contrast, no cell culture system has so far revealed a strong requirement for Vpr in HIV infection, although Vpr-defective HIV-1 shows attenuated replication in human macrophages (2, 6, 16). Despite the absence of a clear defective phenotype of HIV lacking Vpr, this viral Protein has so far attracted more attention than Vpx, presumably because of its intriguing ability to arrest the cell cycle at the G2/M transition (15, 21, 34, 35). We and others recently demonstrated that Vpr recruits DCAF1/VprBP, an adaptor of the CUL4A-DDB1 ubiquitin ligase complex (3, 8, 18, 26, 38, 46). A major role of ubiquitin ligases is the labeling of specific Proteins for proteasome-mediated degradation, and the current hypothesis is that Vpr diverts the DCAF1 ubiquitin ligase to induce the degradation of a host Protein required for entry into mitosis. We previously showed that Vpx from SIVsm also interacts with DCAF1, although unlike Vpr, it does not arrest the cell cycle (26). The ability to recruit DCAF1 might thus represent an ancient functional acquisition that predates the emergence of the genetically related but functionally distinct Vpr and Vpx genes. Here, we explore this hypothesis and address the potential role of Vpx-DCAF1 interaction in the context of HIV-2 replication in human macrophages.
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the human immunodeficiency virus type 2 vpx Protein usurps the cul4a ddb1 dcaf1 ubiquitin ligase to overcome a postentry block in macrophage infection
Journal of Virology, 2009Co-Authors: Anna Bergamaschi, Erwann Le Rouzic, Marina Morel, Diana Ayinde, Annie David, Gilles Collin, Diane DescampsAbstract:Human immunodeficiency virus type 2 (HIV-2), the second causative agent of human AIDS (5), arose from cross-transmission of sooty mangabey simian immunodeficiency virus (SIVsm), a lentivirus that naturally infects sooty mangabeys without inducing overt disease (14). Although AIDS caused by HIV-2 is as lethal as that caused by HIV-1, most HIV-2 carriers remain asymptomatic much longer than HIV-1 carriers (36). On the other hand, cross-transmission of SIVsm to rhesus macaques gave rise to highly pathogenic viral strains, and infected animals develop a disease similar to human AIDS (14). Thus, the SIVsm lentiviral lineage exhibits markedly different pathogenicities in its original and recently acquired hosts. It is currently unknown whether cross-species transmission of primate lentiviruses leads to specific functional changes in viral Proteins that might directly modulate pathogenesis in the new host species. The HIV-1 and HIV-2 lentiviral lineages differ in the auxiliary Proteins encoded by their genomes, which likely reflects different selective pressures in their adaptation to the host cell environment. Thus, the Vpx Protein found in HIV-2 has no counterpart in HIV-1, whereas the genetically related Vpr Protein is present in both lineages (47). Both Vpx and Vpr are actively packaged into the virions (1, 40), which suggests a role in early infection steps, i.e., prior to viral synthesis. Lack of Vpx strongly decreases the pathogenicity of SIVsmPBj in infected macaques (17). In vitro, Vpx is dispensable for infection of immortalized T cells but has been shown to be critical for primary macrophage infection (9, 23). In contrast, no cell culture system has so far revealed a strong requirement for Vpr in HIV infection, although Vpr-defective HIV-1 shows attenuated replication in human macrophages (2, 6, 16). Despite the absence of a clear defective phenotype of HIV lacking Vpr, this viral Protein has so far attracted more attention than Vpx, presumably because of its intriguing ability to arrest the cell cycle at the G2/M transition (15, 21, 34, 35). We and others recently demonstrated that Vpr recruits DCAF1/VprBP, an adaptor of the CUL4A-DDB1 ubiquitin ligase complex (3, 8, 18, 26, 38, 46). A major role of ubiquitin ligases is the labeling of specific Proteins for proteasome-mediated degradation, and the current hypothesis is that Vpr diverts the DCAF1 ubiquitin ligase to induce the degradation of a host Protein required for entry into mitosis. We previously showed that Vpx from SIVsm also interacts with DCAF1, although unlike Vpr, it does not arrest the cell cycle (26). The ability to recruit DCAF1 might thus represent an ancient functional acquisition that predates the emergence of the genetically related but functionally distinct Vpr and Vpx genes. Here, we explore this hypothesis and address the potential role of Vpx-DCAF1 interaction in the context of HIV-2 replication in human macrophages.
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hiv1 Vpr arrests the cell cycle by recruiting dcaf1 Vprbp a receptor of the cul4 ddb1 ubiquitin ligase
Cell Cycle, 2007Co-Authors: Erwann Le Rouzic, Marina Morel, Nadia Belaidouni, E Estrabaud, Jeanchristophe Rain, Catherine Transy, Florence MargottingoguetAbstract:How the HIV1 Vpr Protein initiates the host cell response leading to cell cycle arrest in G2 has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA‑mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G2 arrest by hijacking the Cul4/DDB1DCAF1 ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related Protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both Proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.
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the Vpr Protein from hiv 1 distinct roles along the viral life cycle
Retrovirology, 2005Co-Authors: Erwann Le Rouzic, Serge BenichouAbstract:The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, Vpr, vpx and vpu. While the tat and rev genes encode regulatory Proteins absolutely required for virus replication, nef, vif, Vpr, vpx and vpu encode for small Proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary Proteins are essential for viral replication and pathogenesis in vivo. The two Vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single Vpr gene. In this review, we will mainly focus on Vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle.
Tomoshige Kino - One of the best experts on this subject based on the ideXlab platform.
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human immunodeficiency virus hiv 1 viral Protein r suppresses transcriptional activity of peroxisome proliferator activated receptor γ and inhibits adipocyte differentiation implications for hiv associated lipodystrophy
Molecular Endocrinology, 2008Co-Authors: Shashi Shrivastav, Ulrich Schubert, Tomoshige Kino, Tshaka Cunningham, Takamasa Ichijo, Peter Heinklein, George P Chrousos, Jeffrey B. KoppAbstract:HIV-1-infected patients may develop lipodystrophy and insulin resistance. We investigated the effect of the HIV-1 accessory Protein viral Protein R (Vpr) on the activity of the peroxisome proliferator-activating receptor-γ (PPARγ), a key regulator of adipocyte differentiation and tissue insulin sensitivity. We studied expression of PPARγ-responsive reporter genes in 3T3-L1 mouse adipocytes. We investigated Vpr interaction with the PPAR/retinoid X receptor (RXR)-binding site of the c-Cbl-associating Protein (CAP) gene using the chromatin immunoprecipitation assay as well as the interaction of Vpr and PPARγ using coimmunoprecipitation. Finally, we studied the ability of exogenous Vpr Protein to enter cultured adipocytes and retard differentiation. We found that Vpr suppressed PPARγ-induced transactivation in both undifferentiated and differentiated 3T3-L1 cells. Transcriptional suppression by Vpr required an intact LXXLL coactivator motif. Vpr suppressed mRNA expression of PPARγ-responsive genes in undiffer...
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Vpr Protein of human immunodeficiency virus type 1 binds to 14 3 3 Proteins and facilitates complex formation with cdc25c implications for cell cycle arrest
Journal of Virology, 2005Co-Authors: Tomoshige Kino, George P Chrousos, Alexander Gragerov, Antonio Valentin, Maria Tsopanomihalou, Galina Ilyinagragerova, Rebecca Erwincohen, George N PavlakisAbstract:Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 Proteins, a family regulating a multitude of Proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 Proteins regulate the G 2 /M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 Proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G 2 /M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter9s phosphorylation status.
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human immunodeficiency virus type 1 accessory Protein Vpr a causative agent of the aids related insulin resistance lipodystrophy syndrome
Annals of the New York Academy of Sciences, 2004Co-Authors: Tomoshige Kino, George P ChrousosAbstract:: Recent advances in the development of three different types of antiviral drugs, the nucleotide and non-nucleotide analogues acting as reverse transcriptase inhibitors (NRTIs) and the nonpeptidic viral protease inhibitors (PI), and their introduction in the management of patients with AIDS, either alone or in combination, have dramatically improved the clinical course of the disease and prolonged life expectancy in patients with AIDS. The increase in life expectancy in association with the long-term use of the above antiviral agents, however, have generated novel morbidities and complications. Central among them is the quite common AIDS-related insulin resistance and lipodystrophy syndrome, which is characterized by a striking phenotype and marked metabolic disturbances. To look for the pathologic causes of this particular syndrome, we focused on one of the HIV-1 accessory Proteins, Vpr, which has multiple functions, such as virion incorporation, nuclear translocation of the HIV-1 preintegration complex, nucleo-cytoplasmic shuttling, transcriptional activation, and induction of apoptosis. Vpr may also act like a hormone, which is secreted into the extracellular space and affects the function of distant organs. Vpr functions as a coactivator of the glucocorticoid receptor and potentiates the action of glucocorticoid hormones, thereby inducing tissue glucocorticoid hypersensitivity. Vpr also arrests host cells at the G2/M phase of the cell cycle by interacting with novel 14-3-3 Proteins. Vpr facilitates the interaction of 14-3-3 and its partner Protein Cdc25C, which is critical for the transition of G2/M checkpoint in the cell cycle, and suppresses its activity by segregating it into the cytoplasm. The same Vpr Protein also suppresses the association of 14-3-3 with other partner molecules, the Foxo transcription factors. Since the Foxo Proteins function as negative transcription factors for insulin, Vpr may cause resistance of tissues to insulin. Through these two newly identified functions of Vpr, namely, coactivation of glucocorticoid receptor activity and inhibition of insulin effects on Foxo Proteins, Vpr may participate in the development of AIDS-related insulin resistance/lipodystrophy syndrome.
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hiv 1 Protein Vpr suppresses il 12 production from human monocytes by enhancing glucocorticoid action potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in hiv 1 infection
Journal of Immunology, 2002Co-Authors: Marco Mirani, George P Chrousos, Ilia Elenkov, Simona Volpi, Naoki Hiroi, Tomoshige KinoAbstract:The HIV-1 Protein Vpr has glucocorticoid receptor coactivator activity, potently increasing the sensitivity of glucocorticoid target tissues to cortisol. Patients with AIDS and normal cortisol secretion have manifestations compatible with glucocorticoid hypersensitivity of the immune system, such as suppression of innate and cellular immunities. The latter can be explained by glucocorticoid-induced inhibition of cytokine networks regulating innate and Th1-driven cellular immunity. We demonstrated that extracellularly administered Vpr Protein dose-dependently potentiated glucocorticoid-induced suppression of both mRNA expression and secretion of IL-12 subunit p35 and IL-12 holo-Protein, but not IL-12 subunit p40 or IL-10, by human monocytes/macrophages stimulated with LPS or heat-killed, formalin-fixed Staphylococcus aureus (Cowan strain 1). This effect was inhibited by the glucocorticoid receptor antagonist RU 486. Also, Vpr changed the expression of an additional five glucocorticoid-responsive genes in the same direction as dexamethasone and was active in potentiating the trans-activation, but not the trans-repression, properties of the glucocorticoid receptor on nuclear factor κB- or activating Protein 1-regulated simple promoters. Thus, extracellular Vpr enhances the suppressive actions of the ligand-activated glucocorticoid receptor on IL-12 secretion by human monocytes/macrophages. Through this effect, Vpr may contribute to the suppression of innate and cellular immunities of HIV-1-infected individuals and AIDS patients.
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human immunodeficiency virus type 1 hiv 1 accessory Protein Vpr induces transcription of the hiv 1 and glucocorticoid responsive promoters by binding directly to p300 cbp coactivators
Journal of Virology, 2002Co-Authors: Tomoshige Kino, George P Chrousos, Alexander Gragerov, Olga Slobodskaya, Maria Tsopanomichalou, George N PavlakisAbstract:The accessory Vpr Protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting Protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A Protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators.
Eric A Cohen - One of the best experts on this subject based on the ideXlab platform.
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the human immunodeficiency virus type 1 Vpr Protein upregulates pvr via activation of the atr mediated dna damage response pathway
Journal of General Virology, 2013Co-Authors: Lia Vassena, Erica Giuliani, Giulia Matusali, Eric A Cohen, Margherita DoriaAbstract:Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr Protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same Protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the Protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4+ T lymphocytes and inhibits the PVR downregulating activity of the viral Nef Protein.
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deregulation of micrornas by hiv 1 Vpr Protein leads to the development of neurocognitive disorders
Journal of Biological Chemistry, 2013Co-Authors: Ruma Mukerjee, Eric A Cohen, Robert J Chang, Luis Del Valle, Asen Bagashev, Monika M Gayed, Randolph B Lyde, Brian J Hawkins, Eugen Brailoiu, Christopher PowerAbstract:Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular Proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral Protein R (Vpr), a Protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory Proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr Proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.
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hiv 1 Vpr causes neuronal apoptosis and in vivo neurodegeneration
The Journal of Neuroscience, 2007Co-Authors: Gareth J Jones, Peter Dickie, Eric A Cohen, Nicola Barsby, Janet Holden, Kim H Harris, Jack H Jhamandas, Chris PowerAbstract:Despite the introduction of highly active antiretroviral therapy, dementia caused by human immunodeficiency virus-1 (HIV-1) infection remains a devastating and common neurological disorder. Although the mechanisms governing neurodegeneration during HIV-1 infection remain uncertain, the HIV-1 accessory Protein, viral Protein R (Vpr), has been proposed as a neurotoxic Protein. Herein, we report that Vpr Protein and transcript were present in the brains of HIV-infected persons. Moreover, soluble Vpr caused neuronal apoptosis, involving cytochrome c extravasation, p53 induction, and activation of caspase-9 while exerting a depressive effect on whole-cell currents in neurons ( p p p p p p p p p in vivo neurodegeneration, yielding new insights into the mechanisms by which HIV-1 injures the nervous system.
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human immunodeficiency virus type 1 Vpr mediated g2 cell cycle arrest Vpr interferes with cell cycle signaling cascades by interacting with the b subunit of serine threonine Protein phosphatase 2a
The EMBO Journal, 2000Co-Authors: Mohammed Hrimech, Xiaojian Yao, Philip E Branton, Eric A CohenAbstract:The Vpr Protein of primate lentiviruses arrests cell cycling at the G2/M phase through an inactivation of cyclin B–p34cdc2 and its upstream regulator cdc25. We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 (HIV-1) Vpr mediates G2 arrest by forming a complex with Protein phosphatase 2A (PP2A), an upstream regulator of cdc25. Vpr associates with PP2A through a specific interaction with the B55 regulatory subunit. This interaction is necessary but not sufficient for G2 arrest. Interestingly, we found that Vpr association with B55-containing PP2A targets the enzymatic complex to the nucleus and, importantly, enhances the recruitment and dephosphorylation of the cdc25 substrate. Our data suggest that Vpr mediates G2 arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25.
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human immunodeficiency virus type 1 Vpr Protein transactivation function mechanism and identification of domains involved
Journal of Molecular Biology, 1998Co-Authors: Janique Forget, Xiaojian Yao, Johanne Mercier, Eric A CohenAbstract:The human immunodeficiency virus type 1 (HIV-1) Vpr Protein is a virion-associated Protein that localizes in the nucleus of infected cells. Vpr has been shown to facilitate HIV infection of non-dividing cells such as macrophages by contributing to the nuclear translocation of the pre-integration complex. More recently, Vpr expression has been shown to induce an accumulation of cells at the G2 phase of the cell-cycle. We have previously reported that Vpr stimulates reporter gene expression directed from the HIV-1 long terminal repeat (LTR) as well as from heterologous viral promoters. However, the mode of action of Vpr-mediated transactivation remains to be precisely defined. We report here that, for a constant amount of transfected DNA, the level of chloramphenicol acetyltransferase (CAT) mRNA is increased in Vpr-expressing cells using either HIV-1 or a murine leukemia virus (MLV) SL3-3 LTR-CAT reporter construct. Moreover, this Vpr-mediated transactivation requires that promoters direct a minimal level of basal expression. Our mutagenic analysis indicates that the transactivation mediated by Vpr is not dependent on the ability of the Protein to localize in the nucleus or to be packaged in the virions. Interestingly, all transactivation-competent Vpr mutants were still able to induce a cell-cycle arrest. Conversely, transactivation-defective mutants lost the ability to mediate cell-cycle arrest, implying a functional relationship between these two functions. Overall, our results indicate that the G2 cell-cycle arrest mediated by Vpr creates a cellular environment where the HIV-1 LTR is transcriptionally more active.
Lee Ratner - One of the best experts on this subject based on the ideXlab platform.
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conserved amino acids of the human immunodeficiency virus type 2 vpx nuclear localization signal are critical for nuclear targeting of the viral preintegration complex in non dividing cells
Virology, 2006Co-Authors: Michael Belshan, Lisa A Mahnke, Lee RatnerAbstract:The HIV-2 viral accessory Protein Vpx is related to, but distinct from the Vpr Protein of HIV-1. Vpx is packaged into virions and as a component of the viral preintegration complex (PIC) is required for efficient virus replication in non-dividing cells. We have previously reported that the minimal transferable region of Vpx that contained karyophilic properties was aa 65 to 72. Analysis of Vpx sequences from various HIV-2/SIV strains reveals that this region contains highly conserved amino acids, including two basic residues (K68, R70) and three tyrosines (Y66, Y69, Y71). Here, we demonstrate that mutation of the basic or tyrosine residues abolishes PIC nuclear import in arrested cells as assessed by PCR detection of viral integration. Examination of cell-free virus by Western blot indicated that all mutant Proteins were incorporated into virions, suggesting that the lack of replication in arrested cells was not due to a loss of Vpx in target cells. Together, these studies map critical residues of the Vpx nuclear localization signal that are required for efficient infection of non-dividing cells.
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identification of the nuclear localization signal of human immunodeficiency virus type 2 vpx
Virology, 2003Co-Authors: Michael Belshan, Lee RatnerAbstract:Abstract The Vpx Protein of human immunodeficiency virus type 2 (HIV-2) is a viral accessory Protein related to, but distinct from, the Vpr Protein of HIV-1. Vpx is packaged into virions and, as a component of the viral preintegration complex (PIC), Vpx is required for efficient virus replication in nondividing cells. Therefore, the localization of Vpx in cells is dynamic and dependent upon discrete domains of the Protein. Expressed in the absence of other viral Proteins, Vpx localizes to the nucleus of cells. However, if expressed with the Gag Protein of HIV-2, Vpx localizes to the plasma membrane of cells. To further understand the regulation of Vpx localization, we fused regions of Vpx to β-galactosidase to identify regions of the Protein sufficient to mediate nuclear localization. The minimal transferable region of Vpx that conferred nuclear localization in these assays was aa 65 to 72. Alanine substitution of K68 and R70 in a GFP-Vpx construct abolished nuclear localization, suggesting that the basic residues in this region are important for nuclear import. Analysis of the membrane transport of several GFP-Vpx alanine mutants demonstrated that while separable, the domains of Vpx required for nuclear localization are not distinct from the domains required for membrane transport. The results of heterokaryon shuttling assays indicated that Vpx is not a shuttling Protein; however, HIV-2 Vpr did shuttle similar to HIV-1 Vpr.
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the Vpr Protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Nina Heinzinger, Lee Ratner, Michael Bukrinsky, Sheryl Haggerty, Anna M Ragland, Vineet N Kewalramani, Howard Eliot Gendelman, Mario Stevenson, Michael EmermanAbstract:Abstract The replication of human immunodeficiency virus type 1 (HIV-1) in nondividing host cells such as those of macrophage lineage is an important feature of AIDS pathogenesis. The pattern of HIV-1 replication is dictated, in part, by the nucleophilic property of the viral gag matrix (MA) Protein, a component of the viral preintegration complex that facilitates nuclear localization of viral nucleic acids in the absence of mitosis. We now identify the accessory viral Protein Vpr, as a second nucleophilic component that influences nuclear localization of viral nucleic acids in nondividing cells. Reverse transcription and nuclear localization of viral nucleic acids following infection of cells by viruses lacking Vpr or viruses containing mutations in a gag MA nuclear localization sequence were indistinguishable from the pattern observed in cells infected by wild-type HIV-1. These viruses retained the ability to replicate in both dividing and nondividing host cells including monocyte-derived macrophages. In contrast, introduction of both gag MA and Vpr mutations in HIV-1 attenuated nuclear localization of viral nucleic acids in nondividing cells and virus replication in monocyte-derived macrophages. These studies demonstrate redundant nucleophilic determinants of HIV-1 that independently permit nuclear localization of viral nucleic acids and virus replication in nondividing cells such as monocyte-derived macrophages. In addition, these studies provide a defined function for an accessory gene product of HIV-1.
George P Chrousos - One of the best experts on this subject based on the ideXlab platform.
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human immunodeficiency virus hiv 1 viral Protein r suppresses transcriptional activity of peroxisome proliferator activated receptor γ and inhibits adipocyte differentiation implications for hiv associated lipodystrophy
Molecular Endocrinology, 2008Co-Authors: Shashi Shrivastav, Ulrich Schubert, Tomoshige Kino, Tshaka Cunningham, Takamasa Ichijo, Peter Heinklein, George P Chrousos, Jeffrey B. KoppAbstract:HIV-1-infected patients may develop lipodystrophy and insulin resistance. We investigated the effect of the HIV-1 accessory Protein viral Protein R (Vpr) on the activity of the peroxisome proliferator-activating receptor-γ (PPARγ), a key regulator of adipocyte differentiation and tissue insulin sensitivity. We studied expression of PPARγ-responsive reporter genes in 3T3-L1 mouse adipocytes. We investigated Vpr interaction with the PPAR/retinoid X receptor (RXR)-binding site of the c-Cbl-associating Protein (CAP) gene using the chromatin immunoprecipitation assay as well as the interaction of Vpr and PPARγ using coimmunoprecipitation. Finally, we studied the ability of exogenous Vpr Protein to enter cultured adipocytes and retard differentiation. We found that Vpr suppressed PPARγ-induced transactivation in both undifferentiated and differentiated 3T3-L1 cells. Transcriptional suppression by Vpr required an intact LXXLL coactivator motif. Vpr suppressed mRNA expression of PPARγ-responsive genes in undiffer...
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Vpr Protein of human immunodeficiency virus type 1 binds to 14 3 3 Proteins and facilitates complex formation with cdc25c implications for cell cycle arrest
Journal of Virology, 2005Co-Authors: Tomoshige Kino, George P Chrousos, Alexander Gragerov, Antonio Valentin, Maria Tsopanomihalou, Galina Ilyinagragerova, Rebecca Erwincohen, George N PavlakisAbstract:Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 Proteins, a family regulating a multitude of Proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 Proteins regulate the G 2 /M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 Proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G 2 /M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter9s phosphorylation status.
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human immunodeficiency virus type 1 accessory Protein Vpr a causative agent of the aids related insulin resistance lipodystrophy syndrome
Annals of the New York Academy of Sciences, 2004Co-Authors: Tomoshige Kino, George P ChrousosAbstract:: Recent advances in the development of three different types of antiviral drugs, the nucleotide and non-nucleotide analogues acting as reverse transcriptase inhibitors (NRTIs) and the nonpeptidic viral protease inhibitors (PI), and their introduction in the management of patients with AIDS, either alone or in combination, have dramatically improved the clinical course of the disease and prolonged life expectancy in patients with AIDS. The increase in life expectancy in association with the long-term use of the above antiviral agents, however, have generated novel morbidities and complications. Central among them is the quite common AIDS-related insulin resistance and lipodystrophy syndrome, which is characterized by a striking phenotype and marked metabolic disturbances. To look for the pathologic causes of this particular syndrome, we focused on one of the HIV-1 accessory Proteins, Vpr, which has multiple functions, such as virion incorporation, nuclear translocation of the HIV-1 preintegration complex, nucleo-cytoplasmic shuttling, transcriptional activation, and induction of apoptosis. Vpr may also act like a hormone, which is secreted into the extracellular space and affects the function of distant organs. Vpr functions as a coactivator of the glucocorticoid receptor and potentiates the action of glucocorticoid hormones, thereby inducing tissue glucocorticoid hypersensitivity. Vpr also arrests host cells at the G2/M phase of the cell cycle by interacting with novel 14-3-3 Proteins. Vpr facilitates the interaction of 14-3-3 and its partner Protein Cdc25C, which is critical for the transition of G2/M checkpoint in the cell cycle, and suppresses its activity by segregating it into the cytoplasm. The same Vpr Protein also suppresses the association of 14-3-3 with other partner molecules, the Foxo transcription factors. Since the Foxo Proteins function as negative transcription factors for insulin, Vpr may cause resistance of tissues to insulin. Through these two newly identified functions of Vpr, namely, coactivation of glucocorticoid receptor activity and inhibition of insulin effects on Foxo Proteins, Vpr may participate in the development of AIDS-related insulin resistance/lipodystrophy syndrome.
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hiv 1 Protein Vpr suppresses il 12 production from human monocytes by enhancing glucocorticoid action potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in hiv 1 infection
Journal of Immunology, 2002Co-Authors: Marco Mirani, George P Chrousos, Ilia Elenkov, Simona Volpi, Naoki Hiroi, Tomoshige KinoAbstract:The HIV-1 Protein Vpr has glucocorticoid receptor coactivator activity, potently increasing the sensitivity of glucocorticoid target tissues to cortisol. Patients with AIDS and normal cortisol secretion have manifestations compatible with glucocorticoid hypersensitivity of the immune system, such as suppression of innate and cellular immunities. The latter can be explained by glucocorticoid-induced inhibition of cytokine networks regulating innate and Th1-driven cellular immunity. We demonstrated that extracellularly administered Vpr Protein dose-dependently potentiated glucocorticoid-induced suppression of both mRNA expression and secretion of IL-12 subunit p35 and IL-12 holo-Protein, but not IL-12 subunit p40 or IL-10, by human monocytes/macrophages stimulated with LPS or heat-killed, formalin-fixed Staphylococcus aureus (Cowan strain 1). This effect was inhibited by the glucocorticoid receptor antagonist RU 486. Also, Vpr changed the expression of an additional five glucocorticoid-responsive genes in the same direction as dexamethasone and was active in potentiating the trans-activation, but not the trans-repression, properties of the glucocorticoid receptor on nuclear factor κB- or activating Protein 1-regulated simple promoters. Thus, extracellular Vpr enhances the suppressive actions of the ligand-activated glucocorticoid receptor on IL-12 secretion by human monocytes/macrophages. Through this effect, Vpr may contribute to the suppression of innate and cellular immunities of HIV-1-infected individuals and AIDS patients.
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human immunodeficiency virus type 1 hiv 1 accessory Protein Vpr induces transcription of the hiv 1 and glucocorticoid responsive promoters by binding directly to p300 cbp coactivators
Journal of Virology, 2002Co-Authors: Tomoshige Kino, George P Chrousos, Alexander Gragerov, Olga Slobodskaya, Maria Tsopanomichalou, George N PavlakisAbstract:The accessory Vpr Protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting Protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A Protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators.
