The long-term goal of this Program is to understand the molecular mechanisms that regulate HIV-1 nuclear gene expression and to use this knowledge as a springboard to identify novel targets for therapeutic intervention. A major barrier in HIV-1 biology is that many of the RNA structures and protein-RNA interactions that regulate gene expression in the nucleus are unknown. These processes appear to be combinatorial in nature, and thus systems-level approaches must be combined with functional biochemistry in order to elucidate molecular events that determine patterns of HIV-1 gene expression. Our team (Al-Hashimi, Bieniasz, Cullen, Hargrove, Kutluay, Rouskin, Swanstrom and Tolbert) will use cross-disciplinary expertise in virology, chemical biology and structural biophysics to determine mechanisms of splicing and transcriptional control. We will build on the successes of CRNA 1.0 that include: 1) mapping for the first time binding sites of specific splicing regulatory proteins on the HIV-1 genome; 2) quantitation of the HIV-1 alternative splicing using a novel next generation sequencing platform; 3) identification of novel cis RNA elements that control HIV-1 splicing and 4) determination of the 3D structures of novel HIV-1 RNA splicing elements both free and bound to a regulatory protein. Motivated by these new discoveries, this project will overcome critical barriers that have stymied pursuit of the understanding of HIV-1 splicing regulation. In CRNA 2.0, we will A) reveal how regulatory protein factors and RNA elements cross talk to establish HIV-1 splicing patterns; B) elucidate 3D structures of RNA and protein-RNA complexes that regulate HIV-1 splicing; C) determine the mechanisms by which RNA structure affects splicing; and D) develop novel strategies to target HIV-1 RNA with small molecule inhibitors.
