Quantitative Analysis of the Endogenous Reverse Transcriptase Reactions of HIV Type 1 Variants with Decreased Susceptibility to Azidothymidine and Nevirapine

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Abstract
A large number of nucleoside analog and nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) have been developed for clinical use. Data confirm that resistant variants of HIV-1 rapidly emerge in response to the selective pressure of treatment with these agents. Detection of drug resistance generally involves detection of specific mutations in the viral genome or demonstrating a failure of the drug to suppress virus replication in culture. We have developed a PCR-based method to quantitatively examine HIV-1 DNA synthesis in vitro in endogenous reverse transcription reactions and tested it as a method to detect resistance to RT inhibitors. Under certain conditions, we were able to distinguish HIV strains with high-level resistance to azidothymidine triphosphate inhibition from sensitive strains. This method was quite useful as an assay to detect resistance to nevirapine, a nonnucleoside RT inhibitor; in reconstruction experiments, nevirapine-resistant virus was detectable when it represented 10 to 25% of the total amount of virus present in reaction mixtures. These data are examined in the light of current models of the mechanisms of action of nucleoside and nonnucleoside RT inhibitors. This assay may be useful for detecting the emergence of drug-resistant HIV-1 variants during therapy.