? Understanding the genetic basis of drug resistance is essential for using genotypic drug resistance testing to help physicians select optimal therapy for their patients, for designing new antiviral drugs, and for monitoring the spread of drug-resistant viruses. We plan to identify patterns of protease, reverse transcriptase (RT), and perhaps envelope, and integrase mutations developing in HIV-1 isolates from persons receiving antiretroviral therapy. We will use these patterns to create panels of prototypical infectious biological and molecular clones with high-level resistance to each drug class to assist scientists investigating the biophysical and biochemical basis of drug resistance. To determine whether drug resistance can be explained solely by mutations in the molecular targets of drug therapy, we will compare the susceptibility of biological clones obtained directly from a patient sample with those of recombinant molecular clones in which only the target of therapy has been contributed by the biological clone. We will also compare the susceptibility of recombinant and non-recombinant biological clones from subtype A, C, D, F, G, A/E, and A/G isolates with common patterns of drug-resistance mutations. To examine the role of newly identified treatment-associated RT and protease mutations, we will compare the susceptibility and replication of pairs of clones that are isogenic except for mutation at the position of interest. We hypothesize that HIV-1 isolates with high-level class resistance consist of complex mixtures of clones with related but distinct patterns of drug-resistance mutations. The extent of drug resistance in such isolates is likely to be lower than the cumulative amount of drug resistance in the clones that comprise the isolate. Drugs that are only partially active against isolates with high-level class resistance may not be effective for salvage therapy because these isolates may harbor subpopulations that will require only one or two additional mutations to become highly cross-resistant to a new drug. We will examine the susceptibility of clones from high-level class-resistant isolates to drugs in pre-clinical and clinical development and will culture these clones in the presence of increasing concentrations of these drugs to select viruses with high level cross-resistance for use in drug development efforts. ? ? ?
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