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Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G 2 /M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

Viral protein R (Vpr) is an accessory gene product of human immunodeficiency virus type 1 (HIV-1) that plays multiple important roles associated with viral replication. Structural studies using NMR have revealed that Vpr consists of three α-helices and contains flexible N- and C-termini. However, the molecular mechanisms associated with Vpr function have not been elucidated. To investigate Vpr multifunctionality, we performed an X-ray crystallographic study of Vpr complexes containing importin-α, a known Vpr binding partner present in host cells. Elucidation of the crystal structure revealed that the flexible C-terminus changes its conformation to a twisted β-turn via an induced-fit mechanism, enabling binding to a minor nuclear localization signal (NLS) site of importin-α. The Vpr C-terminus can also bind with major NLS sites of importin-α in an extended conformation in different ways. These results, which represent the first reported crystallographic analysis of Vpr, demonstrate the multifunctional aspects that enable Vpr interaction with a variety of cellular proteins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin alpha alone, in an importin beta-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin alpha from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin alpha at low levels, whereas the expression of three major importin alpha isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin alpha is required for nuclear import of Vpr. Furthermore, interaction between importin alpha and the N-terminal alpha-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin alpha, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

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.

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.

Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.

Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (alphaH1) and between residues 46 and 74 (alphaH2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-alpha. An in vitro transport assay using a series of derivatives of importin-alpha demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin-alpha through alphaH1 and alphaH2; only the interaction via alphaH1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin-alpha functions as a mediator for the nuclear entry of Vpr.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication in primary CD4+ T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked either the ability to accumulate in the nucleus and induce G2 arrest or the ability to induce apoptosis, which has been shown to occur independently of G2 arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G2 arrest but not the induction of apoptosis by Vpr are important for viral replication in primary CD4+ T cells at both high and low multiplicities of infection. Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary CD4+ T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral nuclear import process during infection in primary CD4+ T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was effectively imported into the nucleus by cytosol from primary CD4+ T cells. This Vpr mutant virus replicated well in primary CD4+ T cells, indicating that cellular factors in primary CD4+ T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1.

Vpr, an accessory protein of HIV, is known to affect viral replication as well as cell growth, differentiation, and apoptosis in vitro. To investigate its pathogenicity in vivo, we have produced mice transgenic for the HIV-1 vpr gene with the CD4 enhancer/promoter. Interestingly, apoptotic death of T lymphocytes was enhanced in those mice, causing marked reduction of T cells in lymphatic organs and peripheral blood. Involvement of Bcl-x, Bax, and Caspase-1, but not of the Fas-Fas ligand system, was suggested in the apoptotic processes. These observations suggest that Vpr is involved in the pathogenesis of T cell depletion in HIV-infected people., (Copyright 2001 Academic Press.)

The HIV-1 accessory gene product Vpr can inhibit cell proliferation via cell cycle arrest at the G(2) phase, and it can induce apoptosis after G(2) arrest. We found recently that C81, a carboxy-terminally truncated form of Vpr, induced apoptosis via G(1) arrest but did not induce G(2) arrest of the cell cycle. Thus, it seemed possible that expression of Vpr in cells might cause apoptosis independently of the ability of Vpr to induce G(2) arrest. We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that does not necessarily require induction of G(2) arrest. First, it was found that the extent of apoptosis reached a maximum even when few cells were arrested at the G(2) phase of the cell cycle and was reduced in inverse proportion to the increased induction of G(2) arrest. Thus, the extent of induction of G(2) arrest was not correlated with the extent of Vpr-induced apoptosis. Furthermore, we replaced the Ile/Leu residues in the leucine zipper-like domain of Vpr with Ala or Pro and used cells that expressed the mutant protein to demonstrate that Vpr caused apoptosis in a manner that was independent of G(2) arrest. Finally, replacement of Ile/Leu by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of wild-type Vpr and C81 revealed that the Ile/Leu residues at positions 60, 67, and 74 in the leucine zipper-like domain were indispensable for induction of apoptosis induced by Vpr and by C81 and confirmed, in addition, that both processes might be regulated by the same pathway. C81 appears to be a useful tool for elucidation of the mechanism of apoptosis induced by expression of Vpr protein., (Copyright 2000 Academic Press.)

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within alphaH1 or alphaH2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these alpha-helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the alphaH1 and alphaH2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-alphaH1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-alphaH2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two alpha-helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative alpha-helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G(1) arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1) arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G(2) arrest of the cell cycle.

Vpr, one of the accessory gene products of HIV-1, is a 96-residue protein with several functions. It is involved in import of the HIV-1 preintegration complex into the nucleus of nondividing cells, in cellular differentiation, inducing cell cycle arrest at the G(2)/M phase, in immune suppression, and in enhancement of replication of the virus. We found recently that Vpr interferes with the proliferation of mouse NIH3T3 fibroblasts but fails to arrest these cells in the G(2) phase. Thus, it seems possible that Vpr might retard cell proliferation via a novel pathway that is distinct from G(2) arrest. To elucidate the mechanism by which Vpr induces the retardation of cell growth, we developed a panel of expression vectors that encoded Vpr molecules with deletions of specific putative domains, namely, the first alpha-helical domain, the second alpha-helical domain, a leucine zipper-like domain, and an arginine-rich carboxy-terminal domain. These vectors were introduced into HeLa cells since expression of Vpr can induce G(2) arrest in such cells. A carboxy-terminally truncated form of Vpr, C81, which failed to induce G(2) arrest, led to the G(1) arrest and retained the ability to prevent cell proliferation. All the other mutant proteins had completely lost the capacity to induce G(2) arrest and to suppress growth. Substitutions of Ile/Leu for Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of Vpr or of C81 revealed that Ile60, Leu67, and Ile74 play an important role in the C81-induced suppression of growth, while Ile74 and Ile81 were found to be indispensable for Vpr-induced G(2) arrest. Collectively, our results strongly suggest that Vpr can retard cell proliferation independently of G(2) arrest of the cell cycle., (Copyright 1999 Academic Press.)

Mutations in the human immunodeficiency virus type 1 (HIV-1) vpr gene only slightly reduce the replication rate of the virus. To study the role of HIV-1 Vpr in biological effects on cells, HEp-2 cells, which express HIV-1 Vpr constitutively but at a low level, were established. While control HEp-2 cells underwent apoptosis when incubated with sorbitol, the morphological and biochemical apoptotic changes were inefficiently induced in the HIV-1 Vpr-expressing cells by the same treatment. These results clearly indicate that HIV-1 Vpr has anti-apoptotic activity, and raise the possibility that Vpr acts as a weak activator of virus replication through anti-apoptosis.

Human immunodeficiency virus type 1 Vpr is a 96-amino-acid virion-associated protein that arrests cells in the G2 phase of the cell cycle in peripheral blood lymphocytes, HeLa, 293, 293T, A549, Jurkat, CEM, SupT1, CV-1 and COS1 cells. When we transfected Vpr expression vector into mouse NIH3T3 and then cultured it in the presence of G418, NIH3T3 cells were the drug resistant cells yielded. The surviving colonies, however, exhibited a degenerating morphology up to 8 approximately 20-fold smaller than the control vector colonies. In addition, the growth of NIH3T3 cells transiently transfected with Vpr expression vector declined dramatically compared with that of transfectants with control vector, suggesting that Vpr significantly interferes with cell proliferation of NIH3T3 cells. Cell cycle characterization by flow cytometry indicated that expression of Vpr did not induce G2 cessation in NIH3T3. These findings strongly suggest that Vpr has a novel pathway to retard cell growth independently and arrests the G2 phase of the cell cycle.