Chemical Approaches to Investigate HIV-1 Rev-RRE RNA Interactions



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Two separate crystal structures of the Rev dimer have been solved. One structure features details of the A:A dimerization interface and the other features details of the B:B interface. While the binding of multiple copies of Rev is required for its RNA transport function, the details of the interactions by which Rev interacts with RRE RNA remain elusive. To investigate this, the RWZ2 fragment, which was later named sIIB was fused to a tRNA scaffold and Rev:sIIB-tRNA complexes were analyzed by various biophysical experiments. Establishing the binding strength and stoichiometry in solution has been frustrated by the need for specific chemical labeling of the Rev protein. We have successfully installed a fluorescent probe at the C-terminal end of Rev and demonstrated the binding of RevL with chimeric tRNAPhe-sIIB RNA molecule. We sought to address whether Rev shows any preference in binding (A:A vs. B:B) and dimerization of RNA. We took the approach of making covalently crosslinked Rev dimers in a manner that is consistent with the geometry and conformation of the crystallized Rev dimers. This thesis details the solid phase semisynthetic approach, experimental procedures, and results of the studies done on Rev and the RRE.



Rev, RRE