Mutational Scan of the Human Immunodeficiency Virus Type 2 Integrase Protein

Retroviral integrase (IN) cleaves linear viral DNA specifically near the ends of the DNA (cleavage reaction) and subsequently couples the processed ends to phosphates in the target DNA (integration reaction). In vitro, IN catalyzes the disintegration reaction, which is the reverse of the integration reaction. Ideally, we would like to test the role of each amino acid in the IN protein. We mutagenized human immunodeficiency virus type 2 IN in a random way using PCR mutagenesis and generated a set of mutants in which 35% of all residues were substituted. Mutant proteins were tested for in vitro activity, e.g., site-specific cleavage of viral DNA, integration, and disintegration. Changes in 61 of the 90 proteins investigated showed no phenotypic effect. Substitutions that changed the choice of nucleophile in the cleavage reaction were found. These clustered around the active-site residues Asp-116 and Glu-152. We also found alterations of amino acids that affected cleavage and integration differentially. In addition, we analyzed the disintegration activity of the proteins and found substitutions of amino acids close to the dimer interface that enhanced intermolecular disintegration activity, whereas other catalytic activities were present at wild-type levels. This study shows the feasibility of investigating the role of virtually any amino acid in a protein the size of IN.

逆转录病毒整合酶（Retroviral Integrase, IN）可特异性地在DNA末端附近切割线性病毒DNA（该过程称为切割反应），随后将经加工的DNA末端与靶DNA中的磷酸基团偶联（该过程称为整合反应）。在体外环境中，整合酶可催化解整合反应——整合反应的逆反应。理想状态下，我们希望逐一检测整合酶蛋白中每一个氨基酸残基的功能。本研究采用PCR诱变（PCR mutagenesis）的随机诱变策略，对2型人类免疫缺陷病毒整合酶（human immunodeficiency virus type 2 IN）进行诱变，构建得到一组突变体蛋白，其中全部氨基酸残基中有35%发生了替换。随后对这些突变体蛋白开展体外活性检测，检测指标包括病毒DNA的位点特异性切割、整合反应以及解整合反应。在所检测的90种蛋白变体中，61种发生的氨基酸替换未表现出明显表型效应。研究团队发现，部分替换会改变切割反应中的亲核试剂选择，这类替换的位点集中在活性位点残基天冬氨酸-116（Asp-116）与谷氨酸-152（Glu-152）附近。此外还鉴定出一类氨基酸改变，可对切割与整合过程产生差异化调控效果。本研究同时分析了蛋白的解整合活性，结果显示，位于二聚体界面（dimer interface）附近的氨基酸替换可增强分子间解整合活性（intermolecular disintegration activity），而其余催化活性则维持在野生型水平（wild-type levels）。本研究证实了针对整合酶大小的蛋白质中几乎任意氨基酸残基的功能开展系统性研究的可行性。