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.

HIV-1 depends on host-cell resources for replication, access to which may be limited to a particular phase of the cell cycle. The HIV-encoded proteins Vpr (viral protein R) and Vif (viral infectivity factor) arrest cells in the G 2 phase; however, alteration of other cell-cycle phases has not been reported. We show that Vif drives cells out of G 1 and into the S phase. The effect of Vif on the G 1 -to-S transition is distinct from its effect on G 2 , because G 2 arrest is Cullin5-dependent, whereas the G 1 -to-S progression is Cullin5-independent. Using mass spectrometry, we identified 2 novel cellular partners of Vif, Brd4 and Cdk9, both of which are known to regulate cell-cycle progression. We confirmed the interaction of Vif and Cdk9 by immunoprecipitation and Western blot, and showed that small interfering RNAs (siRNAs) specific for Cdk9 inhibit the Vif-mediated G 1 -to-S transition. These data suggest that Vif regulates early cellcycle progression, with implications for infection and latency. (Blood. 2011;117(4): 1260-1269) IntroductionHIV-1 is the retrovirus that causes AIDS. It carries 6 accessory genes (tat, rev, vif, vpr, vpu, and nef), and 3 structural genes (gag, pol, and env). 1 Vif (viral infectivity factor) of HIV-1 is a 23-kDa phosphoprotein that is expressed late in the retroviral life cycle. Vif is conserved among all of the primate lentiviruses except the equine infectious anemia virus. It is required for HIV-1 infection in primary CD4 T cells, macrophages, and certain T-cell lines (eg, H9 and CEM). [2][3][4][5][6] Viruses lacking a functional vif gene (⌬vif) fail to mount a spreading infection in the above "nonpermissive" cell types. In contrast, many "permissive" T-cell lines (eg, Jurkat and CEMss) and nonhematopoietic cell lines (eg, HeLa and 293T) fully support HIV-1 spreading infection in the absence of Vif. Results from heterokaryon analyses, in which permissive and nonpermissive cell lines have been fused, suggest that the nonpermissive cells express a host-restriction factor that inhibits the replication of HIV-1 virus lacking the vif gene. 7,8 Using a subtractive hybridization approach for analysis of 2 closely related cell lines differing in permissivity, Sheehy et al demonstrated that this host antiviral restriction factor is APOBEC3G (A3G). 9 Vif suppresses the functions of A3G and increases the infectivity of virus produced from infected cells by preventing the packaging of A3G into viral particles. Therefore, Vif plays a vital role in viral replication in primary CD4 T cells both in vitro and in vivo.HIV-1, like all retroviruses, depends on host-cell resources for replication. Access to those resources may be limited to a particular phase of the cell cycle. In human T cells, infection with HIV-1 causes cell-cycle arrest or delay in the G 2 phase of the cell cycle, conferring some advantage to the virus, 10 but leading ultimately to cell death. 11,12 The only viral protein implicated in G 2 arrest of infected cells was Vpr (viral protein R), 11,13-17 until we and others demonstrat...

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.

Groundbreaking research has led to an understanding of some of the pathogenic mechanisms of HIV-1 infection. Surprisingly, an unanswered question remains the mechanism(s) by which HIV-1 inactivates or kills T cells. Our goals are to define candidate T cell signaling cascades altered by HIV infection and to identify mechanisms whereby HIV-infected cells escape the apoptosis triggered by this aberrant signaling. In earlier work, we found that HIV reprograms healthy T cells to self-destruct by a process called apoptosis. We asked whether apoptosis occurs in organs of infected people and made a surprising discovery-this cell death occurs predominantly in healthy bystander cells and only rarely in infected cells. We hypothesize that HIV may be doubly diabolical-healthy T cells are killed in HIV infection, while infected cells resist killing. Thus, the virus protects its viral factory and allows HIV to turn the cell into a "Trojan Horse," with the virus in hiding or "latent." In this review, we discuss the role of viral and cellular proteins in HIV induced T cell anergy and death. We also discuss mechanisms by which HIV may protect infected T cells from apoptosis. These studies will yield new insights into the pathogenesis of AIDS, identify cellular targets that regulate HIV-1 infection, and suggest novel therapeutic approaches to cure HIV infection.

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 G2 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 G2 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 G2 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.