JC virus (JCV) is the etiologic agent of the neurodegenerative disease, progressive multifocal leukoencephalopathy (PML). JCV is normally latent in non-immunocompromised people, but it is activated in brains of individuals with AIDS. Because of the high incidence of PML in AIDS, we have hypothesized that activation of JCV in glial cells of the brain is influenced by HIV-1 infection. We have found that JCV late gene transcription is stimulated by the HIV-1 Tat protein through action at sequence elements bound by the cellular protein, Pur about. Tat and Pur0 about form a highly specific complex, and this complex acts to stimulate transcription at both the HW-1 TAR RNA element and the JCV late promoter. The complex also interacts with T-antigen to enhance JCV DNA replication. Purc about is a frequent partner of cyclin/CDK complexes, as is another Tat-binding protein, cyclin T1. This proposal aims to continue to exploit the expertise of two independent laboratories to coordinate studies on HIV-1, JCV and cellular proteins, including Pur0 about and cyclin T1. We shall take advantage of rapid autopsy procedures at the Manhattan HIV Brain Bank to perform immunogold electron microscopy on PML brain samples to colocalize Tat with these cellular proteins. We shall elucidate the dynamics of the interactions between Tat, T-antigen and Puro about during the course of JCV infection of oligodendrocytes and determine whether transcriptional activation involves activity of cyclin T1/CDK9. We shall characterize the involvement of Purc about or cyclin T1/CDK9 in Tat activation of transcription at TAR(+) or TAR(-) HIV-1 LTR promoters. Use of Puro about point mutants in AA V vectors will help dissect the contributions of different molecular unctional groups to HIV-1 replication in microglial/monocytic cells. We shall pursue our finding that exogenous Tat at low concentrations is incorporated by KG-1 oligodendrocytes and stimulates DNA replication initiated at the JCV origin. We shall determine the mechanism by which Tat enhances replication initiated at the JCV origin in human oligodendrocytes both in vivo and using a new in vitro system. Results will help elucidate pathways of activation of HIV-1 and JCV in the brain and will help target particular molecular interactions for therapy.
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