ABSTRACT
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 G2 phase; however, alteration of other cell-cycle phases has not been reported. We show that Vif drives cells out of G1 and into the S phase. The effect of Vif on the G1- to-S transition is distinct from its effect on G2, because G2 arrest is Cullin5-dependent, whereas the G1- 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 G1- to-S transition. These data suggest that Vif regulates early cell-cycle progression, with implications for infection and latency.
Subject(s)
Cell Cycle/genetics , Cell Proliferation , vif Gene Products, Human Immunodeficiency Virus/physiology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Proliferation/drug effects , Cullin Proteins/genetics , Cullin Proteins/metabolism , Cullin Proteins/physiology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , G1 Phase/drug effects , G1 Phase/genetics , G1 Phase/physiology , Gene Expression Regulation/drug effects , HIV Infections/genetics , HIV Infections/metabolism , HIV Infections/pathology , HIV-1/genetics , HIV-1/physiology , HeLa Cells , Humans , Models, Biological , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding/drug effects , Protein Binding/physiology , RNA, Small Interfering/pharmacology , S Phase/drug effects , S Phase/genetics , S Phase/physiology , Transfection , Virus Latency/drug effects , Virus Latency/genetics , vif Gene Products, Human Immunodeficiency Virus/genetics , vif Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
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.
Subject(s)
G2 Phase , Gene Expression Regulation, Viral , HIV Infections/metabolism , HIV-1/metabolism , vif Gene Products, Human Immunodeficiency Virus/metabolism , vpr Gene Products, Human Immunodeficiency Virus/metabolism , Apoptosis/physiology , Blotting, Western , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Proliferation , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , HIV Infections/pathology , HIV Infections/virology , HIV-1/growth & development , Humans , Leupeptins/pharmacology , Transfection , Virus Replication , vif Gene Products, Human Immunodeficiency Virus/genetics , vpr Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
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.