We propose to study resistance of the human immunodeficiency virus (HIV) to antiviral drugs. Site specific mutagenesis employing linker insertion oligonucleotides will be employed to generate mutation in a cloned HIV reverse transcriptase gene expressed in E. Coli. The mutated RT gene will beassayed for activity and binding to AZT-TP, ddl-TP, PFA and other anti-HIV compounds to correlate mutations with altered RT activity. Mutated RT genes will be inserted back in to the HIV to determine if an altered RT made in vitro will confer resistance of HIV to multiple antiviral drugs. Sensitivity and resistance of clinical isolates of HIV will be assayed by an RNA-RNA hybridization assay employing radiolabelled HIV-RNA probes for the RT gene (pgap) in the presence of increasing concentrations of antiviral will be measured. Patients with AIDS enrolled in of antiviral drugs against HIV will be followed with serial isolations of HIV to assess if sensitivity to antiviral drugs changes with prolonged treatment. The HIV RT gene of initial isolate and subsequent isolate will be amplified by polymerase chain reaction (PCR) to compare the nucleotide sequence of initial and follow-up HIV isolates. If resistance is documented and correlated with base changes in the RT gene, the mutated gene will be inserted into a sensitive train of HIV by recombination to determine if this will confer resistance on a previously sensitive HIV. Patients on antiviral therapy will be followed by direct detection of HIV RNA in blood to assess if the development of resistance to antiviral drugs, particularly AZT, is associated with an increase in viral PNA in eripheral blood. The long term objective of this study is to determine the incidence and frequency of drug resistance developing in HIV in patients receiving antiviral drugs. The mechanisms of resistance will be defined through the use of cloned HIV genes expressed in E.Coli. Through defining the mechanisms of resistance, alternative treatment strategies may be possible for patients with AIDS.
