The overall objective of the proposed research program is to examine the biochemical consequences of the wide genetic diversity in the sequence of the proteolytic enzyme, HIV PR. The primary approach will be the cloning, expression, purification, and kinetic characterization of mutant forms of the enzyme arising from genetic drift in virus populations in HIV-infected individuals. To explore this question, HIV-1 PR variants will be studied with respect to their inhibition by well-characterized proteinase inhibitors. To expand the analysis of the details of active site interactions in the retroviral proteinase class, the following specific aims will be pursued: A. Analysis of genetic variation in HIV-1 PR and the functional consequences. 1. The genetic variability of HIV-1 PR alleles in clinical isolates from periodic samples from individual patients will be identified by amplification and nucleotide sequence analysis of DNA isolated from peripheral blood mononuclear cells of seropositive mothers and their infected children. 2. Analysis of functional activity of variant proteinase molecules will be pursued through expression and careful study with kinetic assays using substrates and inhibitors. A particular objective will be to study potential anti-AIDS drugs, targeted to the proteinase, to determine if the mutant proteinases exhibit resistance to inhibition. 3. Oligonucleotide directed mutagenesis, expression, and purification of selected mutations based on kinetics and structural analysis. Specific hypotheses related to structure-function questions will be studied by the formation of chimeric constructs. B. Analysis of the effect of variation in the sequence of cleavage junctions. 1. The processing of protein fragments containing proteinase and cleavage sites derived from cloned segments will be examined by analyzing processing of expressed protein by SDS-PAGE/Western analysis of a) constructs in which the variant proteinase sequence is fused to a pol gene expression system; b) an expression system in which the variant proteinase is expressed in frame with the upstream gag sequences; c) constructs in which the cleavage sites at the ends of the proteinase are mutated to a non-cleavable form to permit the study of the processing of other cleavage sites within the context of an """"""""extended"""""""" proteinase. 2. Previous studies of cleavage junctions A and B will be expanded to the other known cleavage sites through the synthesis of representative oligopeptides and the study of the rates of cleavage by HIV PR and some of the altered forms developed above. The variations in junction sequences observed in the clones from patient samples will be explored through the synthesis of sets of oligopeptides incorporating these changes, and the rates of cleavage by normal and variant PR will be compared. In addition to providing the foundation for analysis of mutant, chimeric, and other forms of PR, the oligopeptide studies will contribute to understanding, of the mechanisms of precursor processing by further exploration of the question of retroviral proteinase specificity.
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