Tubular-specific expression of HIV protein Vpr leads to severe tubulointerstitial damage accompanied by progressive fibrosis and cystic development.

Chronic kidney disease (CKD) is a common cause of morbidity in human immunodeficiency virus (HIV)-positive individuals. HIV infection leads to a wide spectrum of kidney cell damage, including tubular epithelial cell (TEC) injury. Among the HIV-1 proteins, the pathologic effects of viral protein R (Vpr) are well established and include DNA damage response, cell cycle arrest, and cell death. Several in vitro studies have unraveled the molecular pathways driving the cytopathic effects of Vpr in tubular epithelial cells. However, the in vivo effects of Vpr on tubular injury and CKD pathogenesis have not been thoroughly investigated. Here, we use a novel inducible tubular epithelial cell-specific Vpr transgenic mouse model to show that Vpr expression leads to progressive tubulointerstitial damage, interstitial inflammation and fibrosis, and tubular cyst development. Importantly, Vpr-expressing tubular epithelial cells displayed significant hypertrophy, aberrant cell division, and atrophy; all reminiscent of tubular injuries observed in human HIV-associated nephropathy (HIVAN). Single-cell RNA sequencing analysis revealed the Vpr-mediated transcriptomic responses in specific tubular subsets and highlighted the potential multifaceted role of p53 in the regulation of cell metabolism, proliferation, and death pathways in Vpr-expressing tubular epithelial cells. Thus, our study demonstrates that HIV Vpr expression in tubular cells is sufficient to induce HIVAN-like tubulointerstitial damage and fibrosis, independent of glomerulosclerosis and proteinuria. Additionally, as this new mouse model develops progressive CKD with diffuse fibrosis and kidney failure, it can serve as a useful tool to examine the mechanisms of kidney disease progression and fibrosis in vivo.

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses.

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

A functional conserved intronic G run in HIV-1 intron 3 is critical to counteract APOBEC3G-mediated host restriction.

BACKGROUND: The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat. RESULTS: We identified a G run localized deep in the vpr AUG containing intron 3 (GI3-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of GI3-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of GI3-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by GI3-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of GI3-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in GI3-2, we identified an adjacent G-rich sequence (GI3-1), which was able to substitute for the inactivated GI3-2. CONCLUSIONS: The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.

HIV-1 Vpr stimulates NF-κB and AP-1 signaling by activating TAK1.

BACKGROUND: The Vpr protein of human immunodeficiency virus type 1 (HIV-1) plays an important role in viral replication. It has been reported that Vpr stimulates the nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) signaling pathways, and thereby regulates viral and host cell gene expression. However, the molecular mechanism behind this function of Vpr is not fully understood. RESULTS: Here, we have identified transforming growth factor-ß-activated kinase 1 (TAK1) as the important upstream signaling molecule that Vpr associates with in order to activate NF-κB and AP-1 signaling. HIV-1 virion-associated Vpr is able to stimulate phosphorylation of TAK1. This activity of Vpr depends on its association with TAK1, since the S79A Vpr mutant lost interaction with TAK1 and was unable to activate TAK1. This association allows Vpr to promote the interaction of TAB3 with TAK1 and increase the polyubiquitination of TAK1, which renders TAK1 phosphorylation. In further support of the key role of TAK1 in this function of Vpr, knockdown of endogenous TAK1 significantly attenuated the ability of Vpr to activate NF-κB and AP-1 as well as the ability to stimulate HIV-1 LTR promoter. CONCLUSIONS: HIV-1 Vpr enhances the phosphorylation and polyubiquitination of TAK1, and as a result, activates NF-κB and AP-1 signaling pathways and stimulates HIV-1 LTR promoter.

A novel DCAF1-binding motif required for Vpx-mediated degradation of nuclear SAMHD1 and Vpr-induced G2 arrest.

HIV-2 and closely related SIV Vpx proteins are essential for viral replication in macrophages and dendritic cells. Vpx hijacks DCAF1-DDB1-Cul4 E3 ubiquitin ligase to promote viral replication. DCAF1 is essential for cell proliferation and embryonic development and is responsible for the polyubiquitination of poorly defined cellular proteins. How substrate receptors recruit the DCAF1-containing E3 ubiquitin ligase to induce protein degradation is still poorly understood. Here we identify a highly conserved motif (Wx4Φx2Φx3AΦxH) that is present in diverse Vpx and Vpr proteins of primate lentiviruses. We demonstrate that the Wx4Φx2Φx3AΦxH motif in SIVmac Vpx is required for both the Vpx-DCAF1 interaction and/or Vpx-mediated degradation of SAMHD1. DCAF1-binding defective Vpx mutants also have impaired ability to promote SIVΔVpx virus infection of myeloid cells. Critical amino acids in the Wx4Φx2Φx3AΦxH motif of SIV Vpx that are important for DCAF1 interaction maintained the ability to bind SAMHD1, indicating that the DCAF1 and SAMHD1 interactions involve distinctive interfaces in Vpx. Surprisingly, VpxW24A mutant proteins that were still capable of binding DCAF1 and SAMHD1 lost the ability to induce SAMHD1 degradation, suggesting that Vpx is not a simple linker between the DCAF1-DDB1-Cul4 E3 ubiquitin ligase and its substrate, SAMHD1.VpxW24A maintained the ability to accumulate in the nucleus despite the fact that nuclear, but not cytoplasmic, mutant forms of SAMHD1 were more sensitive to Vpx-mediated degradation. The Wx4Φx2Φx3AΦxH motif in HIV-1 Vpr is also required for the Vpr-DCAF1 interaction and Vpr-induced G2 cell cycle arrest. Thus, our data reveal previously unrecognized functional interactions involved in the assembly of virally hijacked DCAF1-DDB1-based E3 ubiquitin ligase complex.

Simian immunodeficiency virus-specific CD8+ T cells recognize Vpr- and Rev-derived epitopes early after infection.

The kinetics of CD8(+) T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8(+) T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4(+) T cells early after SIV infection.

HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.

Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4(pos) T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4(pos) T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

HIV-1 viral protein R causes peripheral nervous system injury associated with in vivo neuropathic pain.

Painful peripheral neuropathy has become the principal neurological disorder in HIV/AIDS patients. Herein, we investigated the effects of a cytotoxic HIV-1 accessory protein, viral protein R (Vpr), on the peripheral nervous system (PNS). Host and viral gene expression was investigated in peripheral nerves from HIV-infected individuals and in HIV-infected human dorsal root ganglion (DRG) cultures by RT-PCR and immunocytochemistry. Cytosolic calcium ([Ca(2+)]) fluxes and neuronal membrane responses were analyzed in cultured DRGs. Neurobehavioral responses and cytokine levels were assessed in a transgenic mouse model in which the vpr transgene was expressed in an immunodeficient background (vpr/RAG1(-/-)). Vpr transcripts and proteins were detected in peripheral nerves and DRGs from HIV-infected patients. Exposure of rat or human cultured DRG neurons to Vpr rapidly increased [Ca(2+)] and action potential frequency while increasing input resistance. HIV infection of human DRG cultures caused neurite retraction (P<0.05), accompanied by induction of interferon-α (IFN-α) transcripts (P<0.05). vpr/RAG1(-/-) mice expressed Vpr together with increased IFN-α (P<0.05) in the PNS and also exhibited mechanical allodynia, unlike their vpr/RAG1(-/-) littermates (P<0.05). Herein, Vpr caused DRG neuronal damage, likely through cytosolic calcium activation and cytokine perturbation, highlighting Vpr's contribution to HIV-associated peripheral neuropathy and ensuing neuropathic pain.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Viral factors involved in adapter-related protein complex 2 alpha 1 subunit-mediated regulation of human immunodeficiency virus type 1 replication.

The presence of siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) enhances human immunodeficiency virus type 1 (HIV-1) replication by up-regulating nuclear transport of viral genome. In this report, we examined possible viral factors involved in AP2alpha-mediated regulation of HIV-1 replication, namely, Gag matrix protein (MA), integrase (IN) and Vpr. Replication of mutant viruses lacking the nucleophilic property of one of these viral proteins was significantly enhanced by treating cells with AP2alpha siRNA, indicating that Gag MA, IN or Vpr is not specifically involved in AP2alpha-mediated enhancement of viral replication. In contrast, AP2alpha siRNA showed no effect on the level of gene transduction mediated by HIV-1-derived lentiviral vector (LV). Although virus-like LV particle and parental HIV-1 particle are composed of almost equivalent viral structural proteins, LV particles lack three accessory proteins, Vif, Vpr and Vpu, and a large portion of the HIV-1 genome. Vif, Vpr and Vpu were dispensable for AP2alpha siRNA-mediated enhancement of HIV-1 replication, indicating that a particular part of the HIV-1 genomic fragment deleted in the LV genome might be required for the enhancing effect of AP2alpha siRNA on viral replication. Taken together, these results suggest that an as yet undetermined gene fragment of the HIV-1 genome is involved in AP2alpha-mediated regulation of HIV-1 replication.

Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages.

It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr-mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are refractive to the diploid DNA status typical of G2 phase. Our results suggest that the two phenotypes, namely the nuclear localization and the G2-arrest activity of the protein, segregate functionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcription after establishment of an infection. These two functions may render Vpr's role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs.

Genome analysis of small-ruminant lentivirus genotype E: a caprine lentivirus with natural deletions of the dUTPase subunit, vpr-like accessory gene, and 70-base-pair repeat of the U3 region.

The nucleotide sequence of the highly divergent small-ruminant lentivirus genotype E has been determined. The full genome consists of 8,418 nucleotides and lacks two large portions corresponding nearly to the entire dUTPase subunit of the pol and vpr-like accessory genes. Moreover, the 70-bp repeat of the U3 region of the long terminal repeat was observed to be deleted. Interestingly, this lentivirus genotype is able to persist in a local breed population, and retrospective analysis revealed its presence in milk samples collected in 1999. gag sequences obtained from a flock coinfected with the B1 and E genotypes revealed that the evolutionary rates of the two viruses were quite similar. Since a reduced viral load and/or disease progression was observed for viruses with artificially deleted dUTPase and vpr-like genes, it is proposed that this viral cluster be designated a low-pathogenicity caprine lentivirus.

[Anti-glioma effect of combination of bFGF-siRNA and Vpr in nude mice].

OBJECTIVE: To study the anti-glioma effect of recombinant adenovirus mediated combined gene therapy of bFGF-siRNA and HIV1-Vpr in vivo. METHODS: Mouse glioma model was established by injecting 5 × 10(6) LN229 cells into BALB/c-nu nude mice. 30 nude mice were randomly divided into 5 groups: the negative control group, mock group, bFGF-siRNA group, Vpr group and combined therapy group, which at regular intervals were injected with PBS, rAd5-null, rAd5-bFGF-siRNA, rAd5-Vpr, rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volume was recorded every third day to draw a growth curve. After four weeks treatment, the mice were killed and specimens were taken. HE, immunohistochemical and TUNEL staining were performed to observe the cell morphology, detect the changes of relevant target proteins and cell apoptosis, respectively. Also the ultrastructural changes were observed by electron microscopy. RESULTS: The tumor growth inhibition rates were 36.9%, 37.2% and 58.6% in the bFGF-siRNA group, Vpr group and combined therapy group, respectively, and the combined therapy group showed the most significant effect (P < 0.05). Also the results of HE, immunohistochemical and TUNEL staining revealed that the combined therapy group had the best effects on proliferation inhibition and apoptosis induced in glioma cells (P < 0.05). The most significant ultrastructural changes were observed in the combined therapy group. CONCLUSION: The combined gene therapy of bFGF-siRNA with Vpr shows a prominent and synergistic anti-glioma effect compared with that of mono-gene therapy in nude mice.

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins.

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 G2/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.

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.

Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

Visualization of the intracellular behavior of HIV in living cells.

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

Virology. HIV--breaking the rules for nuclear entry.

How does human immunodeficiency virus (HIV) gain access to the carefully guarded nucleus of the host cell? In a Perspective, Segura-Totten and Wilson elaborate on new findings (de Noronha et al.) showing that the HIV protein Vpr is crucial for causing transient herniations in the host cell nuclear envelope. These ruptures are sufficient to enable the preintegration complexes of invading virions to enter the nucleus and to integrate with host cell DNA.

Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.

Adult macaques do not develop disease after infection with a nef deletion mutant of the simian immunodeficiency virus (SIV) and are protected against challenge with pathogenic virus. This finding led to the proposal to use nef-deleted viruses as live, attenuated vaccines to prevent human acquired immunodeficiency syndrome (AIDS). In contrast, neonatal macaques developed persistently high levels of viremia after oral exposure to and SIV nef, vpr, and negative regulatory element (NRE) deletion mutant. Severe hemolytic anemia, thrombocytopenia, and CD4+ T cell depletion were observed, indicating that neither nef nor vpr determine pathogenicity in neonates. Because such constructs have retained their pathogenic potential, they should not be used as candidate live, attenuated virus vaccines against human AIDS.

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.