The primary focus of this proposal is the identification of amino acid interactions that are critical for the formation of stable HIV-1 reverse transcriptase (RT) heterodimers. A detailed understanding of these interactions will potentially provide a basis for the rational design of new classes of therapeutically useful inhibitors for the processes involved in forming an active HIV-1 RT. The proposed experiments will distinguish between interactions critical for 1) protein folding, 2) stability of the folded protein structure, and 3) formation of the p66/p51 heterodimer. Approaches to the identification of peptide inhibitors for various stages in the synthesis and maturation of the RT heterodimer will also be investigated. The experimental approach will rely on efficient methods for production and screening of specific amino acid replacement mutations. The pro-pol-int coding sequence of HIV-1 is expressed in E. coli where it is processed by the encoded protease to produce p66/p51 heterodimer that is indistinguishable from that produced in the infected human cell. Mutant design will be guided by the 3-dimensional X-ray structure of the RT, and by previous mutational analysis which has identified amino acid residues critical for folding and/or stability. To identify interactions critical for dimerization, we have developed a simple assay for stable heterodimers which can be performed on crude extracts of E. coil expressing the RI. The C-terminus of the p66 subunit is tagged with six His residues so that it can be retained on nickel spin columns. The bound RT is eluted, analyzed by Western blotting, and visualized with an anti-RI monoclonal antibody. The wild-type RT shows both p66 and also p51 which is retained on the column in a stable dimer with the His tagged p66. Mutants that destabilize the dimer show little or no bound p51. Mutants at critical residues that produce reduced amounts of correctly folded active RT will be studied to determine whether the residue is critical only during the folding process, or for stability of the correctly folded product. It is expected that residues involved in important steps in the folding process can be identified in this way. Computer modeling of the structures of related RTs (HIV-2, SIV and others) will be performed to study the degree of evolutionary conservation of interactions critical for folding, stability, and dimerization of RT. These RTs will be cloned in our expression system to allow direct study of conserved critical interactions.
