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

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 limited the possibility for new antiviral design targeting these active sites. Therefore, we plan in the project to map the hot spots on the putative PA-PB2 interface by combinatorial alanine-scanning and quantitative saturation mutagenesis in conjunction with high- throughput interaction screening. We aim to dissect the roles played by individual essential residues that make up the binding epitope of PA and PB2. The results obtained in this project are anticipated to improve the molecular understanding of the intrinsic mechanism of virus mRNA transcription as well as to provide opportunities to design the potent inhibitors against influenza virus targeting the key sites on the two polymerase domains.

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。

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。PB1是流感聚合酶异三聚体复合物的核心亚基，负责催化RNA合成，其结构信息最为缺乏。本项目申请人及其合作者计算模建了PB1结构，通过突变该结构上位于RNA模板结合和催化孔道的一系列氨基酸残基，发现了PB1上新的催化关键位点。根据这一区域设计的肽段能抑制流感聚合酶活性，其中PB1(491–515)能够阻断聚合酶与病毒RNA启动子的结合从而抑制流感复制。这一发现增进了对流感聚合酶结构的理解，同时也为针对聚合酶的流感抑制剂研发提供了线索。.同时，本项目申请人及其合作者针对流感病毒蛋白之间以及它们与宿主蛋白之间多存在弱相互作用的特点，建立了偶联微流道系统的无标记椭偏成像蛋白质芯片检测方法。无标记相互作用检测方法因其对蛋白质活性影响小更适合检测弱相互作用或活性难以保持的蛋白质间的相互作用。我们用该方法检测了亲和常数(KA) 约为104 M-1的蛋白质弱相互作用。.另外，快速鉴定流感病毒亚型对病人治疗和流行病防控至关重要，本项目申请人和项目主要参与者建立了采用多重RT-PCR结合DNA膜芯片对流感病毒快速精确分型的方法。