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 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.
Canard
B
B
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Sarfati
S R
SR
Richardson
C C
CC
D003432:Cross-Linking Reagents / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D054306:Dideoxynucleotides; D004351:Drug Resistance / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D054306:Dideoxynucleotides; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III