Structure of Oligomeric HIV Rev-RRE Complexes

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The nuclear export of HIV-1 viral mRNA is mediated by the activity of the regulator Rev. A feature of central importance of Rev function is the ability to self-associate. After Rev binds as a monomer to its primary high affinity RRE-binding site, a combination of cooperative protein-protein and protein-RNA interactions mediate the occupation of secondary binding sites resulting in oligomeric Rev-RRE complexes. Only oligomeric Rev-RRE complexes are nuclear export-competent. This proposal outlines structural studies using liquid state NMR methodology in combination with biochemical approaches that will reveal the detailed nature of the assembly of multiple Rev copies onto the RRE. 1) Characterization of Rev and Rev mutants self-association in the absence and presence of RRE. Rev fibrillizes in solution at concentrations required for liquid state NMR measurements. A screening of buffers facilitating structural analysis of Rev in solution will be performed. We want to identify oligomerization deficiant Rev mutants suitable for high-resolution NMR. We will verify the initial high affinity interaction with RRE stem-loop IIB and will determine stoichiometry of complexes formed by PAGE and ITC assays. 2) Structural Investigation of oligomeric Rev/RRE complexes in solution based on NMR methodology. We plan to solve the structure of Rev dimers providing insight into Rev's strong tendency to self-associate. Structural investigations of different target RRE constructs derived from stem II will be carried out. We will attempt to determine the structure of Rev mutants (monomeric/dimeric) bound to RRE stem-loop IIB and larger RRE variants (stem-loop II). 3) Characterization of structural features of Rev-RRE-inhibitor interactions focussing on ligand structures. We will attempt to determine the structure of the small molecule inhibitors, bound to either Rev and/or RRE using modem NMR methodology. We want to use 5-Fluoropyrimidine substituted RRE RNAs in directed screens utilizing 19F NMR. Based on the three-dimensional structure, we attempt to provide a lead structure for the rational development and synthesis of inhibitors with improved potency.