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.     

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.     

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.     

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.     

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.     

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.     

HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP)--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP) E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.     

Human immunodeficiency virus type 1 Vpr-dependent cell cycle arrest through a mitogen-activated protein kinase signal transduction pathway

The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G(2), which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G(2)/M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.     

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.     

Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as γ-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest.     

Induction of M-phase arrest and apoptosis after HIV-1 Vpr expression through uncoupling of nuclear and centrosomal cycle in HeLa cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces cell cycle arrest in the G2 phase of the cell cycle followed by apoptosis. The mechanism of the arrest is unknown but the arrest is believed to facilitate viral replication. In the present study, we have established cell lines that allow conditional expression of Vpr, and have examined the mechanism of cell death following Vpr expression. We found that cells expressing Vpr enter M phase after long G2 arrest but formed aberrant multipolar spindles that were incapable of completing karyokinesis or cytokinesis. This abnormality provided the basis for apoptosis, which always followed in these cells. The multipolar spindles formed in response to abnormal centrosomal duplication that occurred during the G2 arrest but did not occur in cells arrested in G2 by irradiation. Thus, the expression of Vpr appears to be responsible for abnormal centrosome duplication, which in turn contributes in part to the rapid cell death following HIV-1 infection.     

Dendritic cells infected with vpr-positive human immunodeficiency virus type 1 induce CD8+ T-cell apoptosis via upregulation of tumor necrosis factor alpha

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.     

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.     

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.