The goal of this proposal is to understand how the HIV-1 Tat protein regulates transcription from the HIV-1 LTR. Tat interacts directly with Cyclin T1, the regulatory subunit of CDK9 in the cellular P-TEFb (positive transcription elongation factor) complex. Tat and CycT1 1 bind co-operatively to the viral TAR RNA to recruit P-TEFb to the HIV-1 promoter and facilitate CDK9-mediated phosphorylation of the C-terminal domain (CTD) of the RNAPII large subunit. We recently identified additional proteins that stimulate Tat activity: SKIP (ski-interacting protein), and the GU-alpha RNA helicase II enzyme, which associates with Tat in an RNA-dependent manner in extracts Both proteins enhance HIV-I Tat transcription in vitro and may function during RNAPII elongation. To integrate these and other elongation factors into the overall mechanism of Tat transactivation, we developed a Tat-inducible cell line containing an integrated HIV-1 LTR-eGFP reporter gene that can be used for chromatin immunoprecipitation experiments to examine the precise kinetics and pattern of recruitment of Tat and associated elongation factors to the HIV-1 LTR in vivo.
Five specific aims are proposed here: 1) Identify the protein composition and post-translational modifications of purified native Tat:P-TEFb:TAR complexes. 2) Examine the role of SKIP as a positive effector of HIV-1 transcription by identifying the functional domains required for transcription in vivo and in vitro and identifying cellular proteins that interact with SKIP to stimulate elongation. 3) Examine the role of GU-alpha and other RNA helicases on Tat activity in vivo and in vitro, and effects of RNA helicases on the disassembly of the Tat:P-TEFb:TAR complex during transcription. 4) Characterize additional factors and RNA processing enzymes that bind directly to Tat and/or CycT1. 5) Analyze the Tat-directed recruitment of cellular elongation factors, RNA processing factors, and chromatin remodeling activities at the integrated HIV- 1 LTR promoter by chromatin immunoprecipitation experiments. ? ?
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