The goal of this project is to study the mechanism of cellular control of HIV tat (transactivator) gene function. The focus is on a unique set of human host cell nuclear factors that specifically recognize the stem region of the Tat target sequence TAR RNA. Studies with specific mutants of TAR RNA showed that host factor binding in vitro correlates with Tat function in vivo and that the efficiency of this host cell regulated trans-activation is dependent upon protein kinase C (PKC).
The specific aim of this project is to try to understand the molecular mechanisms that link mitogenic induction of the host cell with viral gene trans-activation. The proposed experiments, using affinity chromatography and immunochemical characterization, are designed to understand the specificity of kinases and phosphatases that regulate functional interaction of the host cell Tat co-factor. Other procedures will attempt molecular cloning and expression of human cDNA sequences encoding the Tat co-factor and study of the mechanism of its regulation of viral activation. Cell-free reconstituted transcription systems will be utilized to decipher the nature of Tat-dependent transcriptional activation of genes under the control of the HIV LTR (long terminal repeat), and the specific role host cell Tat co-factor plays in the regulation of the process. Site-directed mutants of the TAR target sequences and the host cell Tat co-factor will be analyzed in similar reconstituted systems to resolve the nature of their molecular interactions and the functional significance of the host cell modulated viral gene trans-activation.
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|Nekhai, S; Shukla, R R; Fernandez, A et al. (2000) Cell cycle-dependent stimulation of the HIV-1 promoter by Tat-associated CAK activator. Virology 266:246-56|
|Nekhai, S; Shukla, R R; Kumar, A (1997) A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity. J Virol 71:7436-41|