ENHANCED BINDING OF AZIDOTHYMIDINE-RESISTANT HUMAN-IMMUNODEFICIENCY-VIRUS-1 REVERSE-TRANSCRIPTASE TO THE 3'-AZIDO-3'-DEOXYTHYMIDINE 5'-MONOPHOSPHATE-TERMINATED PRIMER

Human immunodeficiency virus type I is resistant to 3'-azido-3'-deoxyt hymidine (AZT) when four amino acid substitutions (D67N, K70R, T215F, and K219Q) are present simultaneously in its reverse transcriptase. Wi ldtype and AZT-resistant reverse transcriptases show identical binding to a 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated p rimer/RNA template. On DNA templates, the equilibrium dissociation con stant (K-D) for primer/template and AZT-resistant reverse transcriptas e (RT) (K-D = 4.1 nM) is similar to that of the wild-type enzyme (K-D = 6.2 nM). However, k(off) is 4-25-fold lower for the AZT-resistant en zyme than for the wild-type enzyme, depending on the nucleotide and th e template. The kinetic decay of a wild-type RT/primer/AZTMP-terminate d DNA template complex is biphasic, Seventy percent of the initial com plex decays with a rate constant greater than 0.05 s(-1), and 30% with a rate constant of 0.0017 s(-1). Decay of an AZT-resistant RT/ AZTMP- terminated primer/DNA template complex is monophasic, with a rate cons tant of 0.0018 s(-1), The Past two nucleotides at the 3' end of the AZ TMP-terminated DNA primer in complex with AZT-resistant RT, but not wi ld-type RT, and a DNA template are protected from exonuclease digestio n, suggesting that enhanced binding of the 3' end of the AZTMP-termina ted DNA primer to reverse transcriptase is involved in the mechanism o f AZT resistance by human immunodeficiency virus type 1.