The long term objective of this multidisciplinary study is to define biological mechanisms involved in retrovirus replication through the study of the biochemical properties of the viral specific protease. These studies are being guided by information obtained from comparison of the crystal structures of the Rous sarcoma virus and the HIV-1 proteases (PRs). In the present studies, the properties of the HIV-1 and RSV PRs will be compared by characterizing their activities on a variety of synthetic peptides and/or truncated precursor polypeptide substrates containing wild type and systematically mutated cleavage sites. These experiments will test structural predictions of enzyme subsites and probe determinants for specific substrate selection and catalytic efficiency. In parallel to the enzymatic studies, site-directed mutagenesis will be used to place specific mutations into the RSV and HIV-1 PRs at amino acid residues that 1) are different between the two enzymes but found in structurally conserved regions, 2) are conserved among retroviral PRs, and 3) are conserved among the related cellular aspartyl proteases. A major strength of this proposal lies in its evaluation of structural differences noted between the HIV-1 and RSV PRs through analysis of bacterially-derived PR molecules that substitute amino acids found in one into the other. A steady state kinetic analysis of each purified mutant PR on homologous and heterologous substrates will be carried out. Furthermore, mutated PR will be placed into both bacterial and viral precursor expression vectors to determine the affect of these mutations on virus replication in vivo.
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