HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT

The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Activation of the ATR-mediated DNA damage response by the HIV-1 viral protein R

DNA damage is a universal inducer of cell cycle arrest at the G2 phase. Infection by the human immunodeficiency virus type 1 (HIV-1) also blocks cellular proliferation at the G2 phase. The HIV-1 accessory gene vpr encodes a conserved 96-amino acid protein (Vpr) that is necessary and sufficient for the HIV-1-induced block of cellular proliferation. In the present study, we examined a recently identified DNA damage-signaling protein, the ATM- and Rad3-related protein, ATR, for its potential role in the induction of G2 arrest by Vpr. We show that inhibition of ATR by pharmacological inhibitors, by expression of the dominant-negative form of ATR, or by RNA interference inhibits Vpr-induced cell cycle arrest. As with DNA damage, activation of ATR by Vpr results in phosphorylation of Chk1. This study provides conclusive evidence of activation of the ATR-initiated DNA damage-signaling pathway by a viral gene product. These observations are important toward understanding how HIV infection promotes cell cycle disruption, cell death, and ultimately, CD4+ lymphocyte depletion.     

Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.

The human immunodeficiency virus type 1 (HIV-1) vpr gene encodes a protein which induces arrest of cells in the G2 phase of the cell cycle. Here, we demonstrate that following the arrest of cells in G2, Vpr induces apoptosis in human fibroblasts, T cells, and primary peripheral blood lymphocytes. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis. However, the alleviation of Vpr-induced G2 arrest by treatment with the drug pentoxifylline did not abrogate apoptosis. Together these studies indicate that induction of G2 arrest, but not necessarily continued arrest in G2, was required for Vpr-induced apoptosis to occur. Finally, Vpr-induced G2 arrest has previously been correlated with inactivation of the Cdc2 kinase. Some models of apoptosis have demonstrated a requirement for active Cdc2 kinase for apoptosis to occur. Here we show that accumulation of the hypophosphorylated or active form of the Cdc2 kinase is not required for Vpr-induced apoptosis. These studies indicate that Vpr is capable of inducing apoptosis, and we propose that both the initial arrest of cells and subsequent apoptosis may contribute to CD4 cell depletion in HIV-1 disease.     

HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells

Human immunodeficiency virus, type 1 (HIV-1), vpr gene encodes a 14-kDa virion-associated protein, which exhibits significant effects on human cells. One important property of Vpr is its ability to induce apoptosis during infection. Apoptotic induction is likely to play a role in the pathogenesis of AIDS. However, the pathway of apoptosis is not clearly defined. In this report we investigate the mechanism of apoptosis induced by HIV-1 Vpr using a Vpr pseudotype viral infection system or adeno delivery of Vpr in primary human lymphoid cells and T-cells. With either vector, HIV-1 Vpr induced cell cycle arrest at the G(2)/M phase and apoptosis in lymphoid target cells. Furthermore, we observed that with both vectors, caspase 9, but not caspase 8, was activated following infection of human peripheral blood mononuclear cell with either Vpr-positive HIV virions or adeno-delivered Vpr. Activation of the caspase 9 pathway resulted in caspase 3 activation and apoptosis in human primary cells. These effects were coincident with the disruption of the mitochondrial transmembrane potential and induction of cytochrome c release by Vpr. The Vpr-induced signaling pathway did not induce CD95 or CD95L expression. Bcl-2 overexpressing cells succumb to Vpr-induced apoptosis. These studies illustrate that Vpr induces a mitochondria-dependent apoptotic pathway that is distinct from apoptosis driven by the Fas-FasL pathway.     

Human immunodeficiency virus type 1 Vpr induces G2 checkpoint activation by interacting with the splicing factor SAP145

Vpr, the viral protein R of human immunodeficiency virus type 1, induces G(2) cell cycle arrest and apoptosis in mammalian cells via ATR (for "ataxia-telangiectasia-mediated and Rad3-related") checkpoint activation. The expression of Vpr induces the formation of the gamma-histone 2A variant X (H2AX) and breast cancer susceptibility protein 1 (BRCA1) nuclear foci, and a C-terminal domain is required for Vpr-induced ATR activation and its nuclear localization. However, the cellular target of Vpr, as well as the mechanism of G(2) checkpoint activation, was unknown. Here we report that Vpr induces checkpoint activation and G(2) arrest by binding to the CUS1 domain of SAP145 and interfering with the functions of the SAP145 and SAP49 proteins, two subunits of the multimeric splicing factor 3b (SF3b). Vpr interacts with and colocalizes with SAP145 through its C-terminal domain in a speckled distribution. The depletion of either SAP145 or SAP49 leads to checkpoint-mediated G(2) cell cycle arrest through the induction of nuclear foci containing gamma-H2AX and BRCA1. In addition, the expression of Vpr excludes SAP49 from the nuclear speckles and inhibits the formation of the SAP145-SAP49 complex. To conclude, these results point out the unexpected roles of the SAP145-SAP49 splicing factors in cell cycle progression and suggest that cellular expression of Vpr induces checkpoint activation and G(2) arrest by interfering with the function of SAP145-SAP49 complex in host cells.     

Human immunodeficiency virus type 1 vpr induces apoptosis through caspase activation

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96-amino-acid protein that is found associated with the HIV-1 virion. Vpr induces cell cycle arrest at the G(2)/M phase of the cell cycle, and this arrest is followed by apoptosis. We examined the mechanism of Vpr-induced apoptosis and found that HIV-1 Vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). We demonstrate that ectopic expression of anti-apoptotic viral proteins, which inhibit caspase activity, and addition of synthetic peptides, which represent caspase cleavage sites, can inhibit Vpr-induced apoptosis. Finally, inhibition of caspase activity and subsequent inhibition of apoptosis results in increased viral expression, suggesting that therapeutic strategies aimed at reducing Vpr-induced apoptosis in vivo require careful consideration.     

Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation

Eukaryotic cells have evolved a complex mechanism for sensing DNA damage during genome replication. Activation of this pathway prevents entry into mitosis to allow for either DNA repair or, in the event of irreparable damage, commitment to apoptosis. Under conditions of replication stress, the damage signal is initiated by the ataxia-telangiectasia-mutated and Rad3-related kinase ATR. We recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) gene product viral protein R (Vpr) arrests infected cells in the G(2) phase via the activation of ATR. In the present study, we show that the activation of ATR by Vpr is analogous to activation by certain genotoxic agents, both mechanistically and in its downstream consequences. Specifically, we show a requirement for Rad17 and Hus1 to induce G(2) arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1. These results demonstrate that G(2) arrest mediated by the HIV-1 gene product Vpr utilizes the cellular signaling pathway whose physiological function is to recognize replication stress. These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4(+)-lymphocyte cell cycle and apoptosis induction in the progressive CD4(+)-lymphocyte depletion characteristic of HIV-1 pathogenesis.     

Anti-vpr activities of heat shock protein 27

HIV-1 Vpr plays a pivotal role in viral pathogenesis and is preferentially targeted by the host immune system. In this report, we demonstrate that a small heat shock protein, HSP27, exhibits Vpr-specific antiviral activity, as its expression is specifically responsive to vpr gene expression and increased levels of HSP27 inhibit Vpr-induced cell cycle G2 arrest and cell killing. We further show that overexpression of HSP27 reduces viral replication in T-lymphocytes in a Vpr-dependent manner. Mechanistically, Vpr triggers HSP27 expression through heat shock factor (HSF) 1, but inhibits prolonged expression of HSP27 under heat-shock conditions. Together, these data suggest a potential dynamic and antagonistic interaction between HIV-1 Vpr and a host cell HSP27, suggesting that HSP27 may contribute to cellular intrinsic immunity against HIV infection.     

Increased levels of Wee-1 kinase in G(2) are necessary for Vpr- and gamma irradiation-induced G(2) arrest

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle arrest at the G(2)/M transition and subsequently apoptosis. Here we examined the potential involvement of Wee-1 in Vpr-induced G(2) arrest. Wee-1 is a cellular protein kinase that inhibits Cdc2 activity, thereby preventing cells from proceeding through mitosis. We previously showed that the levels of Wee-1 correlate with Vpr-mediated apoptosis. Here, we demonstrate that Vpr-induced G(2) arrest correlated with delayed degradation of Wee-1 at G(2)/M. Experimental depletion of Wee-1 by a small interfering RNA directed to wee-1 mRNA alleviated Vpr-induced G(2) arrest and allowed apparently normal progression through M into G(1). Similar results were observed when cells were arrested at G(2) following gamma irradiation. Thus, Wee-1 is integrally involved as a key cellular regulatory protein in the signal transduction pathway for HIV-1 Vpr-induced cell cycle arrest.     

Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis. This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2 arrest and apoptosis of target T cells. A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest. Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress. In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells. Overexpression of Hsp70 reduced the Vpr-dependent G2 arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1. Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1. Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished. Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells. Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr.     

Human immunodeficiency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA alkylating agent, nitrogen mustard.

The product of the human immunodeficiency virus type 1 (HIV-1) vpr gene induces cell cycle arrest in the G2 phase of the cell cycle and is characterized by an accumulation of the hyperphosphorylated form of cdc2 kinase. This phenotype is similar to the effect of DNA-damaging agents, which can also cause cells to arrest at G2. We previously reported that Vpr mimicked some of the effects of a DNA alkylating agent known as nitrogen mustard (HN2). Here we extend these earlier observations by further comparing the activation state of cdc2 kinase, the kinetics of G2 arrest, and the ability to reverse the arrest with chemical compounds known as methylxanthines. Infection of cells synchronized in the G1 phase of the cell cycle with a pseudotyped HIV-1 resulted in arrest at G2 within 12 h postinfection, before the first mitosis. Similar to that induced by HN2, Vpr-induced arrest led to a decrease in cdc2 kinase activity. Vpr-mediated G2 arrest was alleviated by methylxanthines at concentrations similar to those needed to reverse the G2 arrest induced by HN2, and cells proceeded apparently normally through at least one complete cell cycle. These results are consistent with the hypothesis that Vpr induces G2 arrest through pathways that are similar to those utilized by DNA-damaging agents.     

Human immunodeficiency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair.

The human immunodeficiency virus type 1 (HIV-1) vpr gene is an evolutionarily conserved gene among the primate lentiviruses HIV-1, HIV-2, and simian immunodeficiency viruses. One of the unique functions attributed to the vpr gene product is the arrest of cells in the G2 phase of the cell cycle. Here we demonstrate that Vpr interacts physically with HHR23A, one member of an evolutionarily conserved gene family involved in nucleotide excision repair. Interaction of Vpr with HHR23A was initially identified through a yeast two-hybrid screen and was confirmed by the demonstration of direct binding between bacterially expressed recombinant and transiently expressed or chemically synthesized protein products. Visualization of HHR23A and Vpr by indirect immunofluorescence and confocal microscopy indicates that the two proteins colocalize at or about the nuclear membrane. We also map the Vpr-binding domain in HHR23A to a C-terminal 45-amino-acid region of the protein previously shown to have homology to members of the ubiquitination pathway. Overexpression of HHR23A and a truncated derivative which includes the Vpr-binding domain results in a partial alleviation of the G2 arrest induced by Vpr, suggesting that the interaction between Vpr and HHR23A is critical for cell cycle arrest induced by Vpr. These results provide further support for the hypothesis that Vpr interferes with the normal function of a protein or proteins involved in the DNA repair process and, thus, in the transmission of signals that allow cells to transit from the G2 to the M phase of the cell cycle.     

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.     

Suppressive effect of elongation factor 2 on apoptosis induced by HIV-1 viral protein R

Rapid CD4+ lymphocyte depletion due to cell death caused by HIV infection is one of the hallmarks of acquired immunodeficiency syndrome. HIV-1 viral protein R (Vpr) induces apoptosis and is believed to contribute to CD4+ lymphocyte depletion. Thus, identification of cellular factors that potentially counteract this detrimental viral effect will not only help us to understand the molecular action of Vpr but also to design future antiviral therapies. In this report, we describe identification of elongation factor 2 (EF2) as such a cellular factor. Specifically, EF2 protein level is responsive to vpr gene expression; it is able to suppress Vpr-induced apoptosis when it is overproduced beyond its physiological level. EF2 was initially identified through a genome-wide multicopy suppressor search for Vpr-induced apoptosis in a fission yeast model system. Overproduction of fission yeast Ef2 completely abolishes Vpr-induced cell killing in fission yeast. Similarly, overexpression of the human homologue of yeast Ef2 in a neuroblastoma SKN-SH cell line and two CD4+ H9 and CEM-SS T-cell lines also blocked Vpr-induced apoptosis. The anti-apoptotic property of EF2 is demonstrated by its ability to suppress caspase 9 and caspase 3-mediated apoptosis induced by Vpr. In addition, it also reduces cytochrome c release induced by Vpr, staurosporine and TNFα. The fact that overproduction of EF2 blocks Vpr-induced cell death both in fission yeast and human cells, suggested that EF2 posses a highly conserved anti-apoptotic activity. Moreover, the responsive elevation of EF2 to Vpr suggests a possible host innate antiviral response.     

DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest

Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G2 arrest. On the other hand, Vpr-induced G2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest.     

Infection with Vpr-positive human immunodeficiency virus type 1 impairs NK cell function indirectly through cytokine dysregulation of infected target cells

Human immunodeficiency virus type 1 (HIV-1) infection has been implicated in impairing various aspects of NK cell function in viremic condition, and several viral factors contribute to these defects. Here, we evaluated the effect of HIV-1 Vpr on NK cell cytolytic function and cytokine (gamma interferon [IFN-gamma]) production in the context of infection and exposure. Our data indicate that NK cells derived from a peripheral blood mononuclear cell culture infected in vitro with HIV-1 vpr(+) virus or exposed to recombinant Vpr protein exhibited reduced target cell killing in conjunction with diminished expression of CD107a and reduced IFN-gamma production compared to their Vpr-negative counterparts. This Vpr-induced NK cell defect is in part through differential regulation of interleukin-12 and transforming growth factor beta production by the infected target cells and concomitant activation of Smad3 signaling pathway. Collectively, these results illustrate the ability of Vpr to impair NK cell-mediated innate immune functions indirectly by dysregulating multiple cytokines in the infected target cells, thus increasing disease severity and affecting the final outcome in HIV-1 infection.     

Upregulation of survivin by HIV-1 Vpr

The human survivin gene belongs to the family of inhibitor of apoptosis proteins (IAP) and is involved in apoptosis inhibition and regulation of cell division. The survivin gene is the only member of the IAP family whose expression is known to be regulated through the cell cycle. Survivin expression reaches the highest levels during the G₂/M transition and then is rapidly degraded during the G₁ phase. Here we report that the human immunodeficiency virus type 1 (HIV-1) upregulates Survivin expression via survivin promoter transactivation. Vpr, an HIV-1 accessory protein that induces cell cycle arrest in G₂/M, is necessary and sufficient for this effect. Blocking Vpr-induced G₂/M arrest leads to elimination of the survivin promoter transactivation by Vpr. Our results suggest that Survivin may be actively involved in regulating cell viability during HIV-1 infection.     

Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1

ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser(345) phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser(345) phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G(2) arrest and Cdk1-Tyr(15) phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic beta-isoform PP2A(Cbeta) and the A regulatory alpha-isoform PP2A(Aalpha) are involved in the G(2) induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr(15) and Chk1-Ser(345). In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser(345) phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated gammaH2AX-Ser(139) phosphorylation and foci formation, but down-regulation of PP2A(Aalpha) or PP2A(Cbeta) did not decrease gammaH2AX-Ser(139) phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aalpha/Cbeta)-mediated G(2) arrest and Chk1-Ser(345) phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cbeta) or PP2A(Aalpha) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aalpha/Cbeta) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.