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 approximately equal to 12 microM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC50 > 200 microM).
Meyer
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PR
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Matsuura
S E
SE
Mian
A M
AM
So
A G
AG
Scott
W A
WA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D015226:Dinucleoside Phosphates / Q000096:biosynthesis / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D009711:Nucleotides / Q000494:pharmacology*; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*
D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D015226:Dinucleoside Phosphates; D009711:Nucleotides; D013942:Thymine Nucleotides; C010110:2',3'-dideoxyadenosine 5'-phosphate; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D000255:Adenosine Triphosphate; D054303:HIV Reverse Transcriptase