Mechanism by which phosphonoformic acid resistance mutations restore 3 '-azido-3 '-deoxythymidine (AZT) sensitivity to AZT-resistant HIV-1 reverse transcriptase

The development of phosphonoformic acid (PFA) resistance against a backgrou nd of 3'-azido-3'-deoxythymidine (AZT) resistance in human immunodeficiency virus type 1 (HIV-1) restores viral sensitivity to AZT, High level AZT res istance requires multiple mutations (D67N/K70R/T215F/K219Q). In order to ch aracterize the mechanism of PFA resistance-mediated resensitization to AZT, the A114S mutation associated with PFA resistance was introduced into the reverse transcriptase (RT) of both wild type and drug-resistant virus. We p reviously showed that pyrophosphorolytic removal of chain-terminating AZT i s the primary mechanism of the AZT resistance phenotype (Arion, D,, Kaushik , N,, McCormick, S., Borkow, G., and Parniak, M.A. (1998) Biochemistry 37, 15908-15917), Introduction of A114S into the AZT resistance background sign ificantly diminishes both the enhanced pyrophosphorolytic activity and the DNA synthesis processivity associated with the AZT resistant RT, The A114S mutation also alters the nucleotide-dependent phosphorolysis activity assoc iated with AZT resistance. The presence of the A114S mutation therefore sev erely impairs the mutant enzyme's ability to excise chain-terminating AZT, The decrease in phosphorolytic activity of RT conferred by the PFA resistan ce A114S mutation resensitizes AZT-resistant HIV-1 to AZT by allowing the l atter to again function as a chain terminator of viral DNA synthesis. These data further underscore the importance of phosphorolytic removal of chain- terminating AZT as the primary mechanism of HIV-1 AZT resistance.