Transcription is essential for HIV-1 to transcribe its genome and for ttie production of new viral particles to perpetuate the infection. The transcriptional program of HIV-1 relies on Tat, a viral encoded transcriptional activator, which functions by assembling with the transcription elongation factor P-TEFb on nascent viral RNAs to activate the transition between initiation and elongation. Detailed dissection of the steps of Tat assembly into transcription complexes, P-TEFb recruitment and activation, is still incomplete. This Research Plan will use interdisciplinary approaches, such virology, biochemistry/structural biology and genome-wide approaches to uncover new insights into the mechanism of Tat activation and the pathogenesis of AIDS. I will characterize the assembly of HIV-1 transcription preinitiation complexes and define the composition of complexes in their inactive, Tat-activated and Tat-inhibited states. The project will be conducted at UCSF, in the rich community of the Dept of Biochemistry and Biophysics, which has state-of-the-art technologies and a very interactive atmosphere of collaborators with vast experience in fields other than virology, such as in vitro assays, proteomics, network of interactions and structural biology. My career development plan includes receiving training and mentorship in proteomics and structural biology to complete characterization of the assembly of HIV-1 and selected cellular complexes. The work proposed during the mentored phase builds heavily on preliminary data and a set of innovative approaches that led to the proposal of a revised model for the assembly of viral transcription complexes, where Tat and inactive P-TEFb are loaded early in the process. This preliminary data, along with our approach to capture transcription complexes, will be explored to define how Tat assembly and transcription activation occurs. For the independent phase, I will make use of the experience gained in in vitro assays, proteomics and genome-wide approaches to examine the assembly of cellular transcription complexes. I will define 1) the subset of genes that Tat regulates by assembly at their promoters, 2) promoter regulatory elements and complexes composition, and 3) correlation with gene expression profiling based on Tat expression and RNAi of P-TEFb and selected factors.
This project will use innovative in vitro assays and genomic-wide scale approaches to characterize the assembly of HIV-1 Tat and elongation factors into viral and cellular transcription complexes. We aim at uncovering the basis of transcription complex assembly for cell homeostasis control, learning the role of Tat in the pathogenesis of AIDS and helping in the desing of alternative strategies to combat viral replication.