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 background of 3'-azido-3'-deoxythymidine (AZT) resistance in human immunodeficiency virus type 1 (HIV-1) restores viral sensitivity to AZT, High level AZT resistance requires multiple mutations (D67N/K70R/T215F/K219Q). In order to characterize 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 previously showed that pyrophosphorolytic removal of chain-terminating AZT is 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 significantly 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 associated with AZT resistance. The presence of the A114S mutation therefore severely impairs the mutant enzyme's ability to excise chain-terminating AZT, The decrease in phosphorolytic activity of RT conferred by the PFA resistance A114S mutation resensitizes AZT-resistant HIV-1 to AZT by allowing the latter 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.