Enhanced Binding of Azidothymidine-resistant Human Immunodeficiency Virus 1 Reverse Transcriptase to the 3′-Azido-3′-deoxythymidine 5′-Monophosphate-terminated Primer

Abstract

Human immunodeficiency virus type I is resistant to 3'-azido-3'-deoxythymidine (AZT) when four amino acid substitutions (D67N, K70R, T215F, and K219Q) are present simultaneously in its reverse transcriptase. Wildtype and AZT-resistant reverse transcriptases show identical binding to a 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer/RNA template. On DNA templates, the equilibrium dissociation constant (K-D) for primer/template and AZT-resistant reverse transcriptase (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 enzyme than for the wild-type enzyme, depending on the nucleotide and the template. The kinetic decay of a wild-type RT/primer/AZTMP-terminated DNA template complex is biphasic, Seventy percent of the initial complex 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 constant of 0.0018 s(-1), The Past two nucleotides at the 3' end of the AZTMP-terminated DNA primer in complex with AZT-resistant RT, but not wild-type RT, and a DNA template are protected from exonuclease digestion, suggesting that enhanced binding of the 3' end of the AZTMP-terminated DNA primer to reverse transcriptase is involved in the mechanism of AZT resistance by human immunodeficiency virus type 1.