Human immunodeficiency virus-1 (HIV-1) is the etiological agent of acquired immune deficiency syndrome (AIDS). The genome of HIV-1 encodes several novel regulatory proteins. One of these, Tat, plays a pivotal role in the life cycle of HIV-1 by regulating HIV-1 mRNA expression. Tat is an essential protein that increases greatly the expression of genes linked to HIV-1 long terminal repeat (LTR) by stimulating transcription. It does so by binding to a structured RNA element, known as TAR RNA, that is present in the 5' untranslated region of all HIV mRNAs. Studies in vivo in cell culture model systems suggest that Tat is a unique activator protein that both increases transcriptional initiation and stabilizes transcriptional elongation. The precise mechanism of transactivation by Tat remains unclear; however, the effects of Tat are likely to be mediated through a direct or indirect interaction with cellular cofactors. A clear understanding of mechanism of Tat-stimulation may facilitate the development of specific molecular strategies that interfere with HIV-1 gene expression and mitigate the pathobiological consequences of infection. A detailed knowledge of the mechanism of transactivation is likely to emerge from the analysis of a cell free system. At the present time, little is known about cellular factors that might be involved in transactivation. In this proposal, a cell free system would be used to identify and characterize cellular factors that cooperate with Tat in the stimulation of HIV-1 transcription. This goal would be accomplished in three specific aims. The first is to reconstitute basal HIV-1 transcription by fractionating nuclear extracts and to establish a minimal system that supports basal but not Tat-stimulated transcription. The minimal system will then be used to purify and study cellular proteins that are required for Tat-stimulated transcription. Thirdly, cellular proteins that interact with TAR RNA and Tat that are required for Tat- stimulated transcription will be purified and characterized. The identification and characterization of cellular activities that play a role in transactivation by Tat should contribute significantly toward an understanding of the mechanism of Tat action and may provide avenues of therapeutic intervention with viral gene expression.
