Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends
Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends
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DOI：
10.1074/jbc.m510628200
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发表时间：
2006-02-17
影响因子：
4.8
通讯作者：
Leis, J
中科院分区：
文献类型：
--
作者：
Chen, AP;Weber, IT;Leis, J
利用结构和生化数据以及分子动力学模拟，构建了HIV-1整合酶（IN）的四聚体模型，该模型含有U3和U5长末端重复序列（LTR）末端20 bp的DNA。它预测了酶表面能与LTR末端相互作用的氨基酸残基。HIV-1、猿猴肉瘤病毒（SIV）和禽肉瘤病毒（ASV）的独立结构比对预测了这些残基中哪些是独特的。为了确定这些残基是否负责LTR末端的特异性识别，将ASV IN中的氨基酸替换到HIV-1 IN的结构等效位置，并确定嵌合体对3'过程U5 HIV-1或ASV双寡核苷酸的能力。该分析表明，LTRs有多个氨基酸接触，并且在HIV-1 IN的许多类似位置上替换ASV IN氨基酸赋予了部分切割ASV底物的能力，同时失去了切割同源HIV-1底物的能力。改变特异性的HIV-1 IN残基包括Val（72）、Ser（153）、Lys(160)-Ile（161）、Gly(163)-Val（165）和His(171)-Leu（172）。由于与单个氨基酸取代的嵌合体相比，结合这些取代的嵌合体显示出更接近野生型ASV IN的U5 ASV底物切割特异性，因此似乎是与LTRs相互作用的IN的总和决定了特异性。最后，HIV-1 in中的Ser153和Val72残基是细胞中对萘啶羧胺和二酮酸相关抑制剂产生抗性的酶的变化之一。因此，参与识别ltr的氨基酸残基是在耐药性发展过程中发生变化的位置之一。
A tetramer model for HIV-1 integrase ( IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3' process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val(72), Ser(153), Lys(160)-Ile(161), Gly(163)-Val(165), and His(171)-Leu(172). Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser153 and Val72 in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.