The Vpr gene of HIV-1 encodes a 96-amino-acid protein which plays multiple roles in the viral life cycle. Recently, the investigator and others have observed that Vpr inhibits proliferation of CD4 T-lymphocytes by inducing cell cycle arrest in G2. This viral function is conserved in evolution as other primate lentiviruses such as HIV-2, SIVmac, and SIVagm encode related genes which also induce G2 arrest. Induction of G2 arrest constitutes a novel observation with profound implications for the survival and immune function of CD4 T-lymphocytes. The investigator's preliminary investigations indicate that cells arrested in G2 by Vpr contain elevated levels of protein phosphatase 2A (PP2A) activity. Furthermore, immunoprecipitation experiments indicate that Vpr physically associates with PP2A. In view of the above observations he hypothesizes that the viral protein Vpr interacts with PP2A and this interaction causes up-regulation of PP2A. PP2A is known to influence the transition from G2 to mitosis by modulating the activity of Wee 1 and cdc25. Therefore, the potential effect of Vpr on PP2A activity provides a mechanistic explanation for the induction of G2 arrest by Vpr. The studies outlined in this application examine the interaction between viral Vpr and PP2A and further the understanding of mechanisms of cell cycle regulation.
The Specific Aims of this application are: 1. To study how virally encoded HIV-1 Vpr activates PP2A and induces G2 arrest. Delivery of Vpr into the cell via viral infection can be accomplished by two routes: as a virion-bound protein and by de novo expression after viral integration. In addition to Vpr, HIV-1 encodes other accessory genes whose contributions to PP2A activation and G2 arrest are unknown. The experiments proposed in this Aim are designed to dissect various mechanistic aspects of the activation of cellular PP2A and induction of G2 arrest by virally expressed Vpr. 2. To characterize the interaction between HIV-1 Vpr and PP2A structurally and functionally. The investigator will identify particular subunit(s) of PP2A which physically interact with HIV-1 Vpr, and regions of PP2A and Vpr that are essential for binding. In addition, he proposes to study the functional consequences of this binding on the enzymatic activity of PP2A.
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