Structure-Based Studies of RNA Binding Proteins from HIV

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DESCRIPTION (provided by the applicant): A 35-base RNA stem-loop called SL1, located near the 5' end of the HIV-1 genome, mediates several functions crucial for viral assembly, and so is a potential new target for antiviral therapy. By binding the nucleocapsid (NCp7) portion of the HIV Gag protein, SL1 forms part of the packaging signal that targets genomic RNA into virions. Contact between SL1 loops in a pair of H1V RNAs (forming an initial "kissing-loop" complex that then refolds into a linear duplex) also initiates genomic dimerization - a process that is facilitated by NCp7 binding and is essential for full infectivity. All biological activities of SL1 depend on its three-dimensional structure. During the previous funded period, we used nuclear magnetic resonance (NMR) spectroscopy to solve the structure of a truncated, 23-base SL1 derivative in both its kissing-loop and linear forms the first such structures to be solved for any retrovirus. Using heteronuclear labeling and multidimensional NMR, we now propose to solve the corresponding structures of full-length, authentic SL1, which contains additional features that are biologically important. By determining the SL1 monomer, kissing-loop, and linear structures, we seek to understand the remarkable efficiency with which SL1 dimerizes and the chemical features that stabilize each successive conformation. We will also determine the structures and dynamics of complexes formed when HIV NCp7 protein binds each RNA conformer, revealing the exact molecular contacts involved and the mechanism by which NCp7 facilitates refolding of SL1 and other nucleic acids. The validity of our structural insights will then be tested through targeted mutagenesis of SL1 and NCp7. Real-time NMR will be used to dissect the sequence of events that occur at individual bases in SL1 as it dimerizes and then linearizes in vitro. In addition, we will solve the complex formed by NCp7 with a heterologous, monomeric RNA ligand (aptamer) that binds with higher affinity than SL1, to identify features that account for its tight binding and could be exploited in rational drug design. Results of these studies will provide a basis for discovering new antivirals that target the SL1/NCp7 interaction.