Transactivation is a conserved function among primate lentivirus Vpr proteins but is not shared by Vpx.

OBJECTIVE: To investigate the transactivating activity of Vpr proteins from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIVs) on various primate lentivirus long terminal repeats (LTRs), and to determine whether the Vpx proteins shared by HIV-2 and SIV are able to transactivate any HIV or SIV promoter. STUDY DESIGN/METHODS: The vpr and vpx genes of the HIVs and SIVs encode virion-associated proteins, which are implicated in viral replication and pathogenesis. HIV-1 Vpr is involved in the transport of the preintegration complex (PIC) to the nucleus, transactivates the viral LTR, and induces cell cycle arrest. HIV-2 and SIV Vpx proteins share amino acid sequence similarities with Vpr and are involved in PIC translocation into the nucleus but are unable to induce cell cycle arrest. We cloned and expressed the vpr and vpx genes from several primate lentiviruses and tested their transactivating ability on HIV-1, HIV-2, SIVmac and SIVagm LTRs cloned upstream of the CAT reporter gene. RESULTS: All Vpr tested had a transactivating effect on several viral LTRs; however, none of the Vpx proteins showed a detectable transactivating effect. CONCLUSIONS: These results indicate that the transactivating properties of Vpr proteins were conserved throughout evolution in primate lentiviruses, which suggests that they have an important role in virus replication.     

HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.     

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest

The HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1_NL4-3-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest.     

Human immunodeficiency virus (HIV-1) Vpr induced downregulation of NHE1 induces alteration in intracellular pH and loss of ERM complex in target cells

Human immunodeficiency virus type 1 (HIV-1) Vpr is known to dysregulate host cellular functions through its interaction with cellular proteins. Using a protein array we assessed Vpr-mediated differential regulation of host cellular proteins expression. Results demonstrated that Vpr differentially regulated host factors that are involved in functions, such as cell proliferation, differentiation and apoptosis. One of the most highly downregulated proteins attained was the sodium hydrogen exchanger, isoform 1 (NHE1), which showed a significant (60%) decrease in HIV-1 Vpr(+) virus infected cells as compared to HIV-1 Vpr(-) virus infected control. NHE1 downregulation further led to acidification of cells and was directly correlated with loss of ezrin, radixin and moesin (ERM) protein complex and decreased AKT phosphorylation. Vpr-mediated NHE1 dyregulation is in part through GR pathway as GR antagonist, mifepristone reversed Vpr-induced NHE1 downregulation.     

Development of RD-tat cell lines: use in HIV recombination studies

The transactivator (tat) gene of human immunodeficiency virus (HIV) plays an essential role in the replication cycle of HIV. Previous studies have evaluated the extent and mechanistic aspects of tat-mediated transactivation using lymphoid and adherent non-lymphoid cells. We have exploited the transactivation property of the tat gene to achieve high levels of hybrid HIV resulting from recombination between HIV DNAs. For this purpose, we have generated stably transformed human rhabdomyosarcoma (RD) cell lines expressing tat gene product of HIV-1. Functional analysis of the cell lines for the presence of tat protein by transfecting HIV-long terminal repeat (LTR) linked to chloramphenicol acetyl transferase (CAT) revealed low, moderate, and high tat producer cell lines. RD-tat cell lines also showed enhanced virus production upon transfection of HIV-1 proviral DNA. Further, tat producer cell lines showed a high amount of hybrid virus in comparison to the control RD cells upon transfection of truncated viral DNAs. Thus, RD-tat cell lines would be valuable target cells for generating homogeneous viruses upon transfection of viral DNA.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

The Vif accessory protein alters the cell cycle of human immunodeficiency virus type 1 infected cells

The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G(2) phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G(2) phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G(2) arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.     

A novel inducible expression system to study transdominant mutants of HIV-1 Vpr

In this study, an episomal system for ecdysone-inducible gene expression was developed. Human embryonic kidney 293 cells (293VE) expressing a heterodimer of modified ecdysone and retinoid X receptors and the Epstein-Barr nuclear antigen-1 were screened. Plasmids containing the EBV replication origin, oriP, and the ecdysone-response element could replicate and persist in 293VE cells to inducibly express luciferase or Vpr. The induction level, tested with luciferase reporter plasmid, varied among cell lines from 254- to 2056-fold. In one highly inducible cell line, HIV-1 Vpr was expressed well and caused G2 cell cycle arrest in the presence of the inducer, while in the absence of the inducer, no Vpr protein or cell cycle arrest could be detected. Using different selection markers, HIV-1 Vpr was coexpressed with Vpr mutants defective in phosphorylation at Ser79 and G2 cell cycle arrest activity. These Vpr mutants were transdominant to wild-type Vpr for G2 cell cycle arrest activity, but did not alter wild-type Vpr phosphorylation. It is likely that the transdominant mutants and wild-type Vpr compete for a downstream target(s) of G2 cell cycle arrest.