A Mechanism of AZT Resistance: An Increase in Nucleotide-Dependent Primer Unblocking by Mutant HIV-1 Reverse Transcriptase

Mutations in HIV-1 reverse transcriptase (RT) give rise to 3′-azido-3′-deoxythymidine (AZT) resistance by a mechanism that has not been previously reproduced in vitro. We show that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type. Although mutant RT also unblocked ddAMP-terminated primers more efficiently than wild-type RT, the removal of ddAMP was effectively inhibited by the next complementary dNTP (IC50 ≈ 12 μM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC50 > 200 μM).