Mutational patterns associated with the 69 insertion complex in multi-drug-resistant HIV-1 reverse transcriptase that confer increased excision activity and high-level resistance to zidovudine
Mutational patterns associated with the 69 insertion complex in multi-drug-resistant HIV-1 reverse transcriptase that confer increased excision activity and high-level resistance to zidovudine
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DOI：
10.1016/j.jmb.2006.09.073
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发表时间：
2007-01-12
影响因子：
5.6
通讯作者：
Menendez-Arias, Luis
中科院分区：
文献类型：
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作者：
Cases-Gonzalez, Clara E.;Franco, Sandra;Menendez-Arias, Luis
人类免疫缺陷病毒I型(HIV-1)毒株对齐多夫定(AZT)和其他核苷类似物表现出高水平的耐药性，这些毒株在FING亚区插入二肽，并在其逆转录酶(RT)编码区散布着其他与耐药性相关的突变。这些表型效应与它们在链终止的引物上增加的依赖于ATP的磷酸化活性有关。突变T69S和T215Y以及在第69和70位之间插入二肽(即Ser-Ser)是实现对胸苷类似物的低水平耐药所必需的。然而，更多的氨基酸替换是必要的，以实现对AZT的高水平表型耐药，这是由在其RT编码区携带二肽的临床分离株所显示的。为了在HIV临床分离株含插入RT(简称SS RT)的序列背景下鉴定导致抗性的突变，我们表达并纯化了一系列包含部分野生型或SS RT序列的嵌合酶。使用AZT和stavudine(D4T)终止的引物进行的ATP介导的切割活性测量和表型分析表明，对AZT的高水平抗性的分子决定因素位于聚合酶的Finger亚区。进一步的研究表明，M41L、A62V和K70R是关键突变，与T69S、T215Y和二肽插入一起，使AZT和d4T末端的引物具有高水平的ATP依赖的磷酸化活性。切割活性与AZT敏感性测定有很好的相关性，并与对d4T的表型抗性一致。结构分析表明，M41L和A62V在3-4发夹环的定位上存在协同作用，在抗性机制中起关键作用。(C)2006爱思唯尔有限公司。保留所有权利。
Human immunodeficiency virus type I (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the 3-4 hairpin loop, which plays a key role in the resistance mechanism. (c) 2006 Elsevier Ltd. All rights reserved.