E92A是HIV-1整合酶耐药突变N155S的活性回复突变

HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.

E92A Is an Activity Recovery Mutation of HIV-1 Integrase Drug Resistance Mutation N155S

HIV-1 integrase (IN) is an important drug target of current anti-AIDS research and drug development. The drug resistance mutations (DRMs) are the main reason of integrase inhibitor therapy failure, but the drug resistance mechanism remains unclear. We introduced mutations artificially into HIV-1 integrase, tested the activity and drug resistance of individual mutations, and analyzed the integrase drug resistance mechanism preliminarily. The mutations involved contain two single mutations, E92A and N155S, and a double mutation, E92A/N155S. These mutations were obtained using genetic engineering methods, and these recombinant proteins were prepared after prokaryotic expression and protein purification. The strand-transfer activity of the integrases was tested by a magnetic beads based ELISA. S-1360 and Raltegravir were used for drug resistance testing. Besides, molecular docking was performed to study the complex of S-1360 and HIV-1 integrase central catalytic domain (including wild type and mutated type) using Autodock. The main results are as follows: N155S mutation decreases about 80% of the integrase strand transfer activity, while E92A/N155S only decreases about 42%. It indicates that, the E92A mutation on the base of N155S mutation increased the integrase activity observably. Besides, E92A and E92A/N155S mutations exhibit different drug resistance to different inhibitors, and they are more resistant to Raltegravir than to S-1360. A mutation could cause structural change of the integrase catalytic center domain, and the structural change eventually influences the activity and drug resistance. As to E92A, it may reduce the electrostatic interaction to the amino acids around it, and influences D64 and D116 indirectly, which are key amino acids of catalytic center domain. This may give a rational explanation to activity recovery of N155S caused by E92A.