This proposal has four specific aims.
Aim 1 will examine the influence of genetic background on the replication and infectivity of virus by replacing a region of the gag-pol gene with segments of varying sequences cloned from HIV-1 infected patients. Virus growth in culture will be quantitated in the absence and presence of relevant drugs.
Aim 2 will study the self-processing of variant Gag-Pol polyproteins derived from patient samples after insertion into a Gag-Pol expression construct. Cleavage by the imbedded protease at six junctions within the 110 kDa mini polyprotein precursor will be studied by changes of intermediate protein species during expression in E. coli using SDS-PAGE and Western blotting. The precursor will also be prepared by over-expression and refolding for processing studies under controlled conditions with and without inhibitors for structural analysis.
Aim 3 will expand studies of the catalytic potential and susceptibility to inhibitors of variant HIV-1 protease species derived by subcloning the protease coding region of the gag/pol segments from patients. Assays using sets of peptides which harbor two or more cleavage sites are proposed. Inhibitor binding will be quantitated and Vitality values will be calculated.
Aim 4 will extend sequence analysis to samples from a pediatric clinical trial of the efficacy of anti-protease drugs. The sequence of HIV protease and cleavage sites from patients will be determined before starting therapy, and at selected points within the protocol. identification of new sequence variants, especially those that develop as a consequence of drug challenge will provide samples to be studied as part of Specific Aims 1, 2 and 3.
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