Background Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes. Results We purified HIV-1 IN 1F 1–212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN 1–212 . Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strand-transfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers. Conclusions The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

中文翻译：

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氨基末端序列对HIV整合酶结构和功能的影响

背景 抗逆转录病毒疗法 (ART) 可以降低由人类免疫缺陷病毒 (HIV) 引起的发病率和死亡率。ART 的成功开发可以通过关于目标及其对抑制剂的反应的准确结构和生化数据来加速。一个重要的 ART 靶标 HIV 整合酶 (IN) 历来以适应细菌过度表达的修饰形式进行体外研究，在 N 端具有甲硫氨酸或更长的融合蛋白序列。相比之下，病毒颗粒中的 IN 是通过 Pol 多蛋白的蛋白水解裂解产生的，在 N 端留下苯丙氨酸 (IN 1F)。对可用结构的检查表明，在 N 端添加残基可能会破坏正确的蛋白质折叠和多聚体复合物的形成。结果 我们纯化了 HIV-1 IN 1F 1-212 并以 2.4 Å 分辨率解析了其结构，与已发表的 IN 1-212 结构相比，这显示了 N 端螺旋的延伸。与含有额外 N 末端残基的两个 IN 变体相比，全长 IN 1F 在两个病毒 DNA 末端的偶联连接分析中显示出更高的体外催化活性。IN 1F 对抑制剂的敏感性也发生了改变，显示对链转移抑制剂 raltegravir 的敏感性降低，而对变构整合酶抑制剂的敏感性增加。在溶液中，IN 1F 以单体和二聚体形式存在，与其他以高阶低聚物形式存在的 IN 制剂相反。结论结构、生化、IN 1F 的生物物理表征揭示了天然 HIV-1 IN N 端的构象以及伴随着独特的生化和生物物理特性。因此，IN 1F 代表了一种改进的试剂，用于体外整合反应和抗逆转录病毒药物的开发。