Individuals infected with the human immunodeficiency virus, HIV-1, frequently develop the neurodegenerative disease, progressive multifocal leukoencephalopathy (PML). PML is caused by the polyomavirus, JCV, a virus normally latent in non-immunocompromised people. Activation of JCV in glial cells of AIDS individuals is thought to be a consequence of HIV- 1 infection. We have found that stimulation of JCV late-gene transcription by the HIV-1 Tat protein is mediated by a cis-acting Tat- responsive element, upTAR, containing recognition sites for the cellular single-stranded DNA-binding protein Puralpha. Puralpha also binds to RNA recognition sites in the HIV-1 TAR element. Furthermore, Puralpha forms a complex with Tat which can be detected by co-immunoprecipitation from extracts of glial cells. The Tat-Puralpha interaction differentially affects transcription initiated at HIV-1 and JCV late gene promoters. This proposal aims to capitalize on the expertise of two independent laboratories to coordinate studies on HIV-I, JCV and Puralpha. We shall determine whether Tat transactivation of JCV late-gene transcription is dependent upon interaction of Tat with Puralpha. We shall assess the ability of Tat and Puralpha to affect transcription at both the HIV-1 promoter and the JCV late-gene promoter in vitro and in glial cells infected with either HIV-1 or JCV. Using a series of Puralpha deletion mutants we shall determine which regions of Puralpha are involved in binding to Tat. We shall determine whether Tat, Puralpha and TAR form a stable ternary complex and whether such a complex plays a role in transactivation. We shall determine which JCV sequences are bound by Puralpha in vivo in human glial cells in the presence or absence of Tat. Finally, using a series of puralpha-derived synthetic peptides, we shall seek peptides which inhibit Tat binding to Puralpha without affecting Tat-responsive JCV gene transcription or HIV-1 replication. Results will provide information about the interaction of HIV-1 and JCV in human glial cells and will help illuminate a potential target for HIV-1 therapy.
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