Conservation and host specificity of Vpr-mediated cell cycle arrest suggest a fundamental role in primate lentivirus evolution and biology.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents infected cells from passing through mitosis by arresting them in the G2 phase of the cell cycle. Vpr is conserved among all primate lentiviruses, suggesting an important role in the virus life cycle. Moreover, in this study we show that the ability to cause cell cycle arrest is also conserved in Vpr proteins from a wide variety of both tissue culture-passaged and uncultured human (HIV-1 and HIV-2), sooty mangabey (simian immunodeficiency virus SIV(SM)), African green monkey (SIV(AGM)), and Sykes' monkey (SIV(SYK)) isolates. However, this property is cell type specific and appears to depend on the particular primate species from which the cells are derived. SIV(AGM) and SIV(SYK) Vpr proteins are capable of arresting African green monkey cells but are completely inactive in human cells. By contrast, HIV-1, HIV-2, and SIV(SM) Vpr proteins function in both simian and human cell types, although SIV(SM) Vpr functions more efficiently in simian cells than it does in human cells. Neither differential protein stability nor subcellular localization explains the species-specific activities of these proteins. These results thus suggest that Vpr exerts its G2 arrest function by interacting with cellular factors that have evolved differently among the various primate species.     

Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.     

Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.     

Molecular insight into how HIV-1 Vpr protein impairs cell growth through two genetically distinct pathways

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 G(2) 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 G(2) arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate G(2) arrest. We took advantage of the characterization of a subset of G(2) arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G(2) arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G(2) arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G(1) 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.     

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.     

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.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr.

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

HIV1 Vpr Arrests the Cell Cycle by Recruiting DCAF1/VprBP,a Receptor of the Cul4-DDB1 Ubiquitin Ligase

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.     

Human immunodeficiency virus type 1 Vpr interacts with spliceosomal protein SAP145 to mediate cellular pre-mRNA splicing inhibition

Vpr, an accessory gene product of human immunodeficiency virus type 1 (HIV-1), affects both viral and cellular proliferation by mediating long terminal repeat activation, cell cycle arrest at the G2 phase, and apoptosis. We previously found that Vpr plays a novel role as a regulator of pre-mRNA splicing both in vivo and in vitro. However, the cellular target of Vpr, as well as the mechanism of cellular pre-mRNA splicing inhibition by Vpr, is unknown. Here, we show clearly that Vpr inhibits the splicing of cellular pre-mRNA, such as beta-globin pre-mRNA and immunoglobulin (Ig) M pre-mRNA and that the third alpha-helical domain and arginine-rich region are important its ability to inhibit splicing. Additionally, using mutants with specific substitutions in two domains of Vpr, we demonstrated that the interaction between Vpr and SAP145, an essential splicing factor, was indispensable for splicing inhibition. Finally, co-immunoprecipitation and in vitro competitive binding assays indicated that Vpr associates with SAP145 and interferes with SAP145-SAP49 complex formation. Thus, these results suggest that cellular expression of Vpr may block spliceosome assembly by interfering with the function of the SAP145-SAP49 complex in host cells.     

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.     

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.     

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-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.     

A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm)

Vpr is a small accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is specifically incorporated into virions. Members of the HIV-2/SIV(sm)/SIV(mac) lineage of primate lentiviruses also incorporate a related protein designated Vpx. We previously identified a highly conserved L-X-X-L-F sequence near the C terminus of the p6 domain of the Gag precursor as the major virion association motif for HIV-1 Vpr. In the present study, we show that a different leucine-containing motif (D-X-A-X-X-L-L) in the N-terminal half of p6(gag) is required for the incorporation of SIV(mac) Vpx. Similarly, the uptake of SIV(mac) Vpr depended primarily on the D-X-A-X-X-L-L motif. SIV(mac) Vpr was unstable when expressed alone, but its intracellular steady-state levels increased significantly in the presence of wild-type Gag or of the proteasome inhibitor lactacystin. Collectively, our results indicate that the interaction with the Gag precursor via the D-X-A-X-X-L-L motif diverts SIV(mac) Vpr away from the proteasome-degradative pathway. While absent from HIV-1 p6(gag), the D-X-A-X-X-L-L motif is conserved in both the HIV-2/SIV(sm)/SIV(mac) and SIV(agm) lineages of primate lentiviruses. We found that the incorporation of SIV(agm) Vpr, like that of SIV(mac) Vpx, is absolutely dependent on the D-X-A-X-X-L-L motif, while the L-X-X-L-F motif used by HIV-1 Vpr is dispensable. The similar requirements for the incorporation of SIV(mac) Vpx and SIV(agm) Vpr provide support for their proposed common ancestry.