Various mechanisms have been proposed to account for the immune dysfunction in AIDS ultimately leading to loss of CD4+ T-cells, including envelope-mediated syncytium formation, apoptosis and cytokine modulation. Our recent results suggest a novel hypothesis forT-cell dysfunction. We used a human immunodeficiency virus type 1 (HIV-1) bearing a novel cell surface reporter gene, murine thy 1.2, to distinguish infected from uninfected cells in the same population. We show that infected cells, both from cell lines and primary T-cells, are unable to progress normally through the cell cycle and become arrested in the G2/M phase. One of the HIV-1 genes, vpr, has been implicated in a number of functions, including trans-activation, nuclear targeting and re-activation of latent HIV-1. We demonstrate that the arrest of cell growth can be attributed to the vpr gene product of HIV-1, as mutation of this gene allows cell cycle progression of the infected cells. Furthermore, by transient transfection assays, we show that expression of vpr alone will induce cell cycle arrest in SupT1 T-cells and HeLa cells. Using a different reporter system, luciferase, we investigate the consequences of G2 arrest for HIV- 1 replication. Our results show that the HIV-1 provirus is not maintained due to the selective growth disadvantage of infected cells. Mutation of vpr results in maintenance of infection. Thus, expression of vpr prevents the establishment of a chronic HIV-1 infection. These results have two predictions that are relevant to HIV-1 pathogenesis: 1) that HIV-1 infection does not generally lead to a chronic infection with persistence of provirus; and 2) that infected T-cells would be unable to undergo clonal expansion as a result of G2/M arrest, with consequences for immune dysfunction. We propose here to extend our initial findings to develop further insights into its mechanism of action.
The Specific Aims are to: 1. Determine the relationship between the G2/M arrest phenotype and other observed functions of the vpr gene. Structure-function relationships will be investigated through genetic approaches. 2. Compare the functions of vpr-related genes from HIV-1, HIV-2 and simian immunodeficiency virus (SIV) in regards to cell cycle arrest. We will also compare the functions of Vpr with those of the evolutionarily conserved vpx gene.
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