Center for Structural Biology of HIV RNA

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This Project consists of a deep analysis of the structures that form on HIV-1 RNAs in infected cells, the viral and host proteins with which they interact, and the critical roles these structures play in HIV-1 gene expression. The virus uses specific RNA structures to regulate gene expression, but in many cases the actual folded structures of the RNAs and of the larger complexes they form with proteins are not yet known in any detail. We have assembled a powerful team of investigators with the broad range of skills and expertise needed to determine the structures, to define the essential portions of the RNAs needed for biological activity, and to unravel the mechanism of action of the RNA-protein complexes that promote virus gene expression and replication. The steps of the viral life cycle that we propose to examine will include: the synthesis of RNA transcripts by RNA polymerase II elongation, as allowed by the release from arrest mediated by the Tat protein, P-TEFb subunits and the TAR RNA; the alternative splicing of HIV-1 RNAs, as controlled by cis-acting elements at the splice sites of the viral precursor RNA; the selective nuclear export of spliced, partially spliced and unspliced mRNAs as controlled by Rev action at the RRE element and by nuclear pore subunits; and the translation of viral mRNAs, including the role of the 5' cap in determining the fate of the RNAs, and the activity of the RNA hairpin at the site of translational frameshifting in regulating expression of the long Gag-Pro-Pol precursor protein. We will study structures of “naked” RNAs in solution, but also in the context of RNA-protein complexes as they exist in intact infected cells. We will use powerful genetic tools and rapid readouts of gene expression to identify host factors involved in these various processes, mutagenesis and CRISPR-based knockouts to probe the functions of these factors and the details of their interactions with RNA. We have come to appreciate that many of the RNA structures are highly dynamic and consist of a constellation of alternative forms – clearly true in the cases of the TAR element, the 5' cap and 5' UTR sequence that control RNA utilization, the splicing regulatory elements, and the translational frameshift element. Our team will apply advanced methods capable of monitoring these dynamic rearrangements at multiple time scales. The results will break new ground in discovery, design, and optimization of viral inhibitors targeting RNA. We initially will address all these RNA structures and their functions in the context of the actively replicating virus in lytic growth in T cells, readily studied in culture. In addition, we are interested in the distinctive regulation of these steps that occurs in the establishment and maintenance of latency, a state allowing persistence of virus as transcriptionally silent proviruses. We will take advantage of a new model of latency to define changes in the RNA structures and the way they are recognized by the altered proteome of the memory T cell. The multiple investigators in this Project are experienced, highly focused on the issues, and prepared to make rapid progress on each Aim outlined for the Project.