A drug resistance mutation in the inhibitor binding pocket of human immunodeficiency virus type 1 reverse transcriptase impairs DNA synthesis and RNA degradation
A drug resistance mutation in the inhibitor binding pocket of human immunodeficiency virus type 1 reverse transcriptase impairs DNA synthesis and RNA degradation
复制标题
DOI：
10.1021/bi9600308
复制
发表时间：
1996-07-30
期刊：
影响因子：
2.9
通讯作者：
Sharma, SK
中科院分区：
文献类型：
--
作者：
Fan, NS;Rank, KB;Sharma, SK
对（烷基氨基）哌啶-双（杂芳基）哌嗪（AAP-BHAP）U-104489具有抗性的人免疫缺陷病毒型（HIV-1）的IIIB株的选择导致逆转录酶（RT）的残基190（G190 E）处的甘氨酸被谷氨酸取代。AAP-BHAP抗性HIV-1显示出降低的体外复制能力[Olmsted，R.一、等人（1966）J. Virol. 70，3698-3705]。我们在这里报告，G190 E突变的重组异源二聚体HIV-1 RT，野生型RT（G190）或G190 A控制突变体相比，结果在40%和80%的聚合酶和RNase H特异性酶活性，分别减少。一个引物延伸试验，允许确定的DNA延长的G190 E突变体RT上的杂聚HIV-1 gag为基础的RNA模板显示DNA聚合的整体下降。通过G190 E RT相关RNase H消化来自[S-35]poly（rA）的RNA产生的产物的大小分布。聚（dT）是显着不同的G190 A RT和观察到的RT相关的RNA酶H活性的G190 E RT的减少是一致的。当挑战与未标记的底物，G190 E RT是相对nonprocessive相对于DNA合成和RNA降解。可以得出结论，G190 E抗性突变对这两种RT功能的有害影响最有可能与观察到的AAP-BHAP-（U-104489-）抗性HIV-1的复制能力延迟有关。
Selection of the IIIB strain of human immunodeficiency virus type (HIV-1) resistant to the (alkylamino)piperidine-bis(heteroaryl)piperazine (AAP-BHAP) U-104489 results in substitution of a glycine to glutamate at residue 190 (G190E) of reverse transcriptase (RT). The AAP-BHAP resistant HIV-1 displays reduced in vitro replication capacity [Olmsted, R. A., et al. (1966) J. Virol. 70, 3698-3705]. We report here that the G190E mutation in recombinant heterodimeric HIV-1 RT, compared to the wildtype RT (G190) or a G190A control mutant, results in a 40% and 80% reduction in the polymerase and RNase H specific enzymatic activities, respectively. A primer-extension assay that allowed determination of DNA elongation by the G190E mutant RT on a heteropolymeric HIV-1 gag-based RNA template showed an overall decrease in DNA polymerization. The size distribution of products generated by G190E RT-associated RNase H digestion of RNA from [S-35]poly(rA). poly(dT) was markedly distinct from that of the G190A RT and was consistent with the observed reduction in RT-associated RNase H activity of the G190E RT. When challenged with unlabeled substrates, the G190E RT was relatively nonprocessive with respect to DNA synthesis and RNA degradation. It is concluded that the deleterious effect of the G190E resistance mutation on both of these RT functions is most likely involved in the observed retarded replication capacity of the AAP-BHAP-(U-104489-) resistant HIV-1.