Given the current limitations of AIDS treatments, largely directed against the enzymatic activities of RT or protease, there remains a great need to develop alternative therapeutics that target other essential viral activities. Combinations of inhibitors that act on different aspects of the viral life cycle might help to create more potent or long-lasting therapies. Here we focus on developing inhibitors against two essential HIV RNA elements, the RRE and TAR, which bind the Rev and Tat proteins, respectively. Our previous studies have shown that the arginine-rich RNA-binding domain of HIV-I Rev binds as an a-helix to an internal loop of the RRE and that the Tat arginine-rich domains from BIV and JDV, two bovine viruses closely related to HIV, adopt p-hairpin conformations upon binding to the major groove of TAR. The structurally distinctive binding ?pockets? formed in these RNAs provide potential targets for peptide or small molecule inhibitors, and we already have identified peptides from combinatorial libraries that bind to the RRE with even higher affinities than Rev and inhibit Rev function. Our goals in this proposal are to expand our understanding of the structural details of these interactions and to identify more potent inhibitors.
In Aim 1, we will identify tight RRE-binding molecules from combinatorial libraries designed in the context of two types of RNAbinding frameworks, the arginine-rich motif and zinc finger.
In Aim 2, we will determine the NMR structures of high-affinity peptide complexes with TAR and the RRE. One of these utilizes a JDV Tat peptide that can recognize HIV-I TAR in addition to the bovine TAR sites.
In Aim 3, we will test the ability of tight RRE binders isolated from combinatorial screens or designed based on structural information to inhibit Rev function and HIV replication.
In Aim 4, we will develop new mammalian cellbased reporter assays to screen directly for Tat and Rev inhibitors. These studies are expected to advance our understanding of RNA-protein recognition in addition to providing potential new avenues to the design of HIV inhibitors.
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