Structural studies of influenza virus polymerase

描述(申请人提供)：流感病毒可引起高度传染性的急性呼吸道疾病。在美国，流感平均每年与大约36000人死亡和大约46亿美元的直接医疗费用有关，在流感大流行期间发生的生命损失和经济繁荣要大得多。由于流感病毒聚合酶具有几种病毒特异性活性，针对该聚合酶的小分子药物可能提供方便、广泛和低成本的流感保护作用。流感病毒聚合酶由PA、PB1和PB2三种蛋白组成，具有不同寻常的内切酶活性。聚合酶的转录是使用从宿主细胞mRNAs中捕获的带帽的引物来启动的。相比之下，RNA复制不使用引物，但需要核蛋白(NP)的存在。尽管进行了广泛的遗传和生化研究，但人们对活性转录和复制复合体形成的机制细节知之甚少。样品制备困难一直是流感病毒聚合酶结构研究的主要障碍之一。利用一种新的表达策略，研究人员已经纯化、鉴定并制备了大量的异源三聚体流感病毒聚合酶。这项拟议研究的长期目标是了解流感病毒聚合酶、NP、病毒RNA和其他基本成分如何协调它们的功能，以确保帽依赖和帽非依赖的RNA合成。其具体目的是：(1)通过研究五个功能中间体的结构，利用低温EM三维重建来阐明流感病毒聚合酶在RNA转录启动和催化过程中的结构和动力学；(2)利用生化分析和X射线结晶学来确定聚合酶PA的结构及其在RNA合成中的作用；以及(3)通过研究(A)NP结合对异源三聚体聚合酶结构和功能的影响以及(B)RNA结合对NP寡聚体结构的影响来阐明NP在RNA合成中的作用。

DESCRIPTION (provided by applicant): Influenza viruses cause highly contagious, acute respiratory illnesses. In an average year influenza is associated with approximately 36,000 deaths and approximately $4.6 billion direct medical costs in the United States a much greater loss of life and economic prosperity has occurred during influenza pandemics. Because influenza virus polymerase possesses several virus-specific activities, small molecule drugs targeting the polymerase are likely to offer convenient, broad, and low-cost protection against influenza. Influenza virus polymerase consists of three proteins, PA, PB1 and PB2, and it has an unusual endonuclease activity. Transcription by the polymerase is initiated using a capped primer seized from host cellular mRNAs. In contrast, RNA replication does not use a primer but requires the presence of nucleoproteins (NP). Despite extensive genetic and biochemical studies, mechanistic details underlying the formation of active transcription and replication complexes are poorly understood. Difficulty in sample preparation has been one of the major obstacles in studying the structure of influenza virus polymerase. Using a new expression strategy, the investigator has purified, characterized and made preparative amounts of heterotrimeric influenza virus polymerase. The long-term objective of the proposed research is to understand how influenza virus polymerase, NP, the viral RNA, and other essential components coordinate their functions to ensure both cap-dependent and cap-independent RNA synthesis. The specific aims are to: (1) use cryo-EM 3-D reconstruction to elucidate the structure and dynamics of influenza virus polymerase during initiation and catalysis of RNA transcription by studying the structures of five functional intermediates; (2) determine the structure of the polymerase subunit PA and its role in RNA synthesis using biochemical assays and X-ray crystallography; and (3) elucidate the role of NP in RNA synthesis by studying (a) the effects of NP binding on the heterotrimeric polymerase structure and function and (b) the effects of RNA binding on the structure of NP oligomers.