组合丙氨酸扫描突变和定量饱和突变结合大规模相互作用筛选鉴定流感聚合酶PA-PB2亚基相互作用关键位点

项目名称： 组合丙氨酸扫描突变和定量饱和突变结合大规模相互作用筛选鉴定流感聚合酶PA-PB2亚基相互作用关键位点

项目编号： No.31300632

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 张红

作者单位： 中国科学院生物物理研究所

项目金额： 22万元

中文摘要： 流感是各国面临的公共卫生难题。现有流感防控手段有其局限，亟需寻找新型靶点和抑制剂。流感病毒异三聚体RNA聚合酶由PA、PB1和PB2三亚基组成,司职流感RNA基因组复制和转录，因其保守性和在病毒生命周期中的关键作用有望成为潜在广谱抗流感药物靶点。聚合酶通过"帽子捕获"机制转录流感mRNA，PA内切酶结构域和PB2 5'7-甲基鸟苷酸帽子结合结构域与此过程相关。申请人所在课题组计算分析首次发现这两结构域在模拟结构中位置临近。准确界定它们的相互作用界面和界面上关键氨基酸位点有助了解病毒mRNA转录机理并可根据位点信息设计新型流感抑制剂。本项目拟采用组合丙氨酸扫描突变和定量饱和突变结合大规模相互作用筛选，确定这两个功能关联的结构域的相互作用界面，找出其上参与PA-PB2相互作用、偶联转录过程的关键位点。通过阻断该相互作用能够破坏病毒mRNA转录，抑制病毒复制。

中文关键词： 流感病毒；RNA聚合酶；PB1；相互作用；流感分型

英文摘要： Influenza is a worldwide public health concern and new therapeutics that protect against this highly adaptable virus are urgently needed. The viral RNA-dependent RNA polymerase is a heterotrimer of subunits PA, PB1 and PB2. The polymerase catalyzes both the transcription and replication steps of the virus genome that are central to the viral life cycle and represents a promising target for designing new anti-influenza therapeutics due to its activities and conservation. In particular, transcription of viral mRNAs through the cap-snatching mechanism is essential for virus-replication. Inhibition of either the endonuclease activity of the PA N-terminal domain or the cap-binding activity of the PB2 cap-binding domain is potential means to block influenza virus replication. The computational analysis performed in our lab has revealed that the PA endonuclease domain is adjacent to the PB2 cap-binding domain in the modeled structure, suggesting that the cap-binding and cleavage steps of transcription could be coordinated by the interaction of these two domains. Some fragment crystal structures of these two isolated domains have been determined, but how they interact with each other in the polymerase complex is still unclear. The absence of the detailed structural information of the polymerase complex has severely limi

英文关键词： influenza virus；RNA polymerase；PB1；interaction；typing