The Role of Vpr in HIV-1 Disease Progression is Independent of Its G2 Arrest Induction Function

Vpr (viral protein R) is a vital HIV-1 accessory protein with multiple functions in the viral life cycle, including nuclear import of preintegration complex, induction of apoptosis and G2 cell cycle arrest. The cell cycle perturbation activity of Vpr requires activation of the ATR (Ataxia-Telangiectasia and Rad3-related) pathway and the integrity of Vpr C-terminal motif that is crucial for chromatin binding. Recent studies also demonstrated Vpr as one of the viral factors that influence HIV disease progression, as mutations in Vpr were overrepresented in some cohorts of long-term nonprogressors (LTNP). The LTNP-associated mutations of Vpr are frequently observed in the C-terminal domain. This raises the question whether the LTNP phenotype of Vpr is the result of the loss its ability to induce G2 arrest. Here we report that the LTNP-associated mutants of Vpr function normally in the induction of G2 arrest. No defects in ATR activation and direct binding to chromatin are observed. These mutants also show similar levels of apoptosis induction as wild-type Vpr. These data differentiate the LTNP-associated mutations of Vpr with those defective in inducing G2 arrest. We propose that the G2 arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr.     

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.     

The dose-dependent H2O2 stress response promotes increased survival forSchizosaccharomyces pombe cells expressing HIV-1 Vpr

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during infection, including induction of the cell cycle G2 arrest, and cell death in both human cells and the fission yeast Schizosaccharomyces pombe. We show that treament of exponential-phase wild-type Vpr-expressing S. pombe cells with a low, subinhibitory concentration (0.15 mmol/L) of hydrogen peroxide and 0.1 mmol/L thiamine significantly increased both cell proliferation and survival rates and decreased the number of elongated G2-arrested cells. Short-term, H2O2-induced adaptive stress increased the survival of the cells while acute stress conditions interrupted the Vpr-mediated death of the cells; however, no changes in cell length or cell phase were detected. The results suggest the importance of the oxidative status of the cells in Vpr-mediated processes. Our findings contribute to the development of a new approach via which to investigate the contribution of Vpr to HIV pathogenesis and to reduce the Vpr-mediated effects in HIV-infected patients.     

Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways.

Vpr, one of the accessory molecules of HIV-1, has been demonstrated to arrest the cell cycle at the G2 phase. This Vpr-mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr-mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T-cell line-adapted HIV-1SF2 strain (Vpr2) could not significantly induce G2 arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G2 arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species-non-specific growth inhibitory activity of Vpr was not mediated solely by the C-terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G2 arresting activity. In addition, the species-non-specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.     

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.     

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover

Background

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.

Methodology/Principal Findings

We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.

Conclusions/Significance

This work identifies UNG2 and SMUG1 as novel targets for CRL4^DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4^DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4^DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.     

Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2

Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.     

The HIV-1 Vif protein mediates degradation of Vpr and reduces Vpr-induced cell cycle arrest

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.     

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.     

Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo

The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of gamma-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.     

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.     

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.     

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.     

Depletion of Wee-1 kinase is necessary for both human immunodeficiency virus type 1 Vpr- and gamma irradiation-induced apoptosis

Human immunodeficiency virus (HIV) protein R (Vpr) induces G2 arrest, and prolonged G2 arrest leads to apoptosis. We find that in HeLa cells the cell cycle regulatory kinase, Wee-1, is depleted following prolonged G2 arrest induced by Vpr. Of note, small interfering RNAs directed to Wee-1 triggered apoptosis, suggesting a direct role for Wee-1 in apoptosis. In support of this hypothesis, overexpression of Wee-1 suppressed Vpr-mediated apoptosis. Importantly, similar results were observed with cells induced to undergo apoptosis gamma irradiation. Thus, Wee-1 may serve as a key regulator of both HIV type 1 Vpr- and gamma irradiation-mediated apoptosis and possibly serve as a general regulator linking the cell cycle to some pathways of apoptosis.     

Vpr Stimulates Viral Expression and Induces Cell Killing in Human Immunodeficiency Virus Type 1-Infected Dividing Jurkat T Cells

In this study we investigated the effects of Vpr during human immunodeficiency virus (HIV) infection of proliferating Jurkat T cells by using a vesicular stomatitis virus envelope G glycoprotein pseudotyped HIV superinfection system. We observe that the expression of Vpr results in a severe reduction in the life span of HIV type 1 (HIV-1)-infected dividing T cells in culture. In agreement with a recent report (S. A. Stewart, B. Poon, J. B. M. Jowett, and I. S. Chen, J. Virol. 71:5579–5592, 1997), we show that events characteristic of apoptotic cell death are involved in the Vpr-mediated cytopathic effects. Our results also show that infection with viruses expressing the wild-type vpr gene results in an increase in viral gene expression and production. Interestingly, the effects of Vpr on cell viability and on viral gene expression both correlate with the ability of the protein to induce a cell cycle arrest in the G₂/M phase. Mutagenesis analyses show that the C terminus of Vpr is essential for these biological activities. Although the role of Vpr is currently associated with the infection of nondividing cells, our results suggest that Vpr can also directly increase viral replication in vivo in infected dividing T cells. Furthermore, these in vitro observations suggest that Vpr-mediated cytotoxic effects could contribute to the CD4⁺ depletion associated with AIDS progression.