Human immunodeficiency virus (HIV-1), isolated from patients after long-term treatment with 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine), may exhibit as much as 100-fold reduced sensitivity to the drug. This finding is of major concern since many AIDS patients are currently undergoing prolonged AZT therapy. Similar concerns apply to patients treated with other antiviral drugs such as dideoxyinosine (ddl). AZT resistance is associated with specific mutations in the coding sequence for the virus-encoded reverse transcriptase. This proposal focuses on two areas related to reverse transcriptase and drug-resistant HIV. First, we have prepared recombinant HIV-1 reverse transcriptase containing amino acid substitutions at positions 215 and 219 and have observed a 20-fold increase in the IC50 for AZTTP measured in crude extract or in partially purified enzyme. This suggests that the drug resistant phenotype is due to changes that can be detected in enzyme function and we propose to carry out extensive kinetic studies and structural analysis on this mutant protein and on other mutant proteins to define the mechanism of AZT resistance at the molecular level. Effects of AZTTP on polymerase activity will be measured with templates and primers designed to correspond to different subreactions catalyzed by reverse transcriptase in the viral replication cycle. Inhibition of RNase H activity by AZT-containing nucleotides will also be compared for mutant and wild type enzymes. In order to determine the biological significance of these mutations, we will characterize clinical samples from pediatric patients by direct DNA sequence analysis after proviral DNA amplification using the polymerase chain reaction. Each specimen will also be assayed for drug sensitivity by virus culture assay and the level of drug resistance will be related to the specific mutations detected. DNA sequence analysis and drug sensitivity assays will also be carried out on specimens from pregnant women who have been treated with AZT and their infants to test for transmission of mutations associated with drug resistance from mother to infant. The ultimate goal of this research is to understand molecular mechanisms of drug-resistance mutations in HIV and to improve our ability to monitor drug-resistant virus infections.
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