Enhanced binding of azidothymidine-resistant human immunodeficiency virus 1 reverse transcriptase to the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer

J Biol Chem. 1998 Jun 5;273(23):14596-604. doi: 10.1074/jbc.273.23.14596.


Human immunodeficiency virus type 1 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. Wild-type 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 (KD) for primer/template and AZT-resistant reverse transcriptase (RT) (KD = 4.1 nM) is similar to that of the wild-type enzyme (KD = 6.2 nM). However, koff 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 last 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cross-Linking Reagents / metabolism
  • DNA Primers / metabolism
  • Dideoxynucleotides
  • Drug Resistance / genetics*
  • Exodeoxyribonucleases / metabolism
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / enzymology*
  • Humans
  • Kinetics
  • Protein Binding
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / metabolism*
  • Reverse Transcriptase Inhibitors / metabolism
  • Templates, Genetic
  • Thymine Nucleotides / metabolism*
  • Zidovudine / analogs & derivatives*
  • Zidovudine / metabolism
  • Zidovudine / pharmacology*


  • Cross-Linking Reagents
  • DNA Primers
  • Dideoxynucleotides
  • Reverse Transcriptase Inhibitors
  • Thymine Nucleotides
  • 3'-azido-3'-deoxythymidine 5'phosphate
  • Zidovudine
  • zidovudine triphosphate
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III