Molecular characterization of HIV-1 viral protein R (VPR) cell cycle arrest and cytopathicity

Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4(+) T-cell depletion in HIV-1-infected individuals remains unknown. The HIV-1 Vpr accessory protein has been reported to cause cell death, as well as enhance transcriptional activity of the proviral long terminal repeat, target the pre-integration complex to the nucleus, and arrest cells in the G2/M phase. The studies presented in this doctoral thesis tested the hypothesis that Vpr mediates HIV-1 cytopathicity, and aimed to determine the importance of its diverse biological functions in cell killing. Human CD4(+) T-cells were experimentally transfected, infected with virions to deliver Vpr proteins, or infected with HIV-1 encoding mutant Vpr. HIV-1 cytopathicity was significantly reduced in the absence of Vpr, although infected cells still accumulated in G2/M. Vpr was toxic by itself as well, causing necrotic cell death, which correlated with G2/M arrest induction but not nuclear localization or self-association of Vpr. Mutation of hydrophobic residues on the surface of Vpr's third alpha-helix to glutamic acid disrupted all of these properties, implicating this region in multiple Vpr functions. G2/M arrest by Vpr depended in part on the scaffold protein, 14-3-3. Furthermore, biochemical characterization of cell cycle regulatory protein-protein interactions during Vpr G2/M arrest indicated a significant increase in Cdk1, CyclinB1, and Cdc25C binding to 14-3-3, in conjunction with Vpr. However, the impact of whole virus infection on these interactions was less clear, although centrosomal proteins consistently decreased in association with 14-3-3. Surprisingly, the G2/M arrest was not required for HIV-1-infected cell death, as drug-induced G1 blockade could not protect against HIV-1 cytopathicity. Furthermore, blocking infected cells in either G1 or G2/M in trans increased viral protein expression of a cell cycle arrest mutant strain of HIV-1, which correlated with reversion to a cytopathic infection. Together these results indicate that Vpr is a lethal component of HIV-1 and compromises cell viability through both cell cycle dysregulation and enhancement of HIV-1 proviral expression.