AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP.
Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K(M)-values for polymerization but no change in K(D)-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.
Hartl
Maximilian J
MJ
Universität Bayreuth, Lehrstuhl für Struktur und Chemie der Biopolymere & Research Center for Biomacromolecules, 95440 Bayreuth, Germany.
Kretzschmar
Benedikt
B
Frohn
Anne
A
Nowrouzi
Ali
A
Rethwilm
Axel
A
Wöhrl
Birgitta M
BM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D004247:DNA / Q000096:biosynthesis; D054306:Dideoxynucleotides / Q000378:metabolism*; D024882:Drug Resistance, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016415:Sequence Alignment; D016092:Spumavirus / Q000187:drug effects / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*
D054306:Dideoxynucleotides; C034477:RNA primers; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D012313:RNA; C052873:zidovudine triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D012194:RNA-Directed DNA Polymerase