Hartl Maximilian J, Kretzschmar Benedikt, Frohn Anne, Nowrouzi Ali, Rethwilm Axel, Wöhrl Birgitta M
Universität Bayreuth, Lehrstuhl für Struktur und Chemie der Biopolymere & Research Center for Biomacromolecules, 95440 Bayreuth, Germany.
Nucleic Acids Res. 2008 Feb;36(3):1009-16. doi: 10.1093/nar/gkm1087. Epub 2007 Dec 20.
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.
叠氮胸苷（AZT，齐多夫定）是已知的少数几种能抑制泡沫病毒复制的核苷类抑制剂之一。我们之前已经表明，猕猴泡沫病毒（SFVmac）逆转录酶基因中多达四个突变对于赋予对AZT的高抗性是必要的。为了表征AZT抗性的机制，我们在大肠杆菌中表达了两种对AZT具有高度抗性的SFVmac重组逆转录酶，它们的逆转录酶基因含有三个（K211I、S345T、E350K）或四个突变（K211I、I224T、S345T、E350K）。我们的分析表明，这些突变体的聚合活性受损。与HIV-1的AZT抗性逆转录酶不同，SFVmac的AZT抗性酶在动力学性质上存在差异。SFVmac酶对聚（rA）/寡聚（dT）表现出较低的比活性，聚合反应的米氏常数（K(M)）较高，但对DNA/DNA或RNA/DNA底物的解离常数（K(D)）没有变化。突变酶对AZT的抗性基于在ATP存在下切除掺入的抑制剂。四重突变体的额外氨基酸变化似乎对恢复聚合效率很重要。
