Structure-function studies of alpha- and flaviviruses

项目负责人/主要研究者（最后，第一，中间）：Kuhn， Richard J. 2 P01 AI055672-06A1 摘要目前在分子水平上对甲病毒和黄病毒生命周期的理解是不完整的，因为我们缺乏参与RNA复制、基因组包装和颗粒组装的病毒和宿主蛋白的结构基础。这两类病毒对大部分人口构成重大风险，控制感染和疾病的方法很少。因此，我们将继续进行系统的合作研究，为代表这两组病原体的病毒编码蛋白的结构和功能提供比较方法。我们将重点转移到不仅包括病毒蛋白，而且包括宿主蛋白和宿主与病原体之间的适当复合物。为研究这些病毒蛋白的结构和功能而提出的工具是多种多样的和多学科的。我们以前非常重视单个病毒蛋白和完整病毒的X射线晶体学（Michael Rossmann，Jan.et Smith）。除了我们强调晶体学的高分辨率结构外，Tim Baker，Wen Jiang和Michael Rossmann在病毒，受体和抗体的复合物以及组装/拆卸中间体的冷冻电子显微镜研究中发挥了关键作用。还将采用电子断层扫描和免疫电子显微镜（Richard Kuhn、Wen Jiang、Michael Rossmann）作为病毒感染期间诱导和/或利用的亚细胞结构成像的手段。功能生化和遗传分析（Richard Kuhn，Janet Smith）将补充和支持结构研究。定点诱变和蛋白质相互作用的方法，探索发现，或假设，结构确定的基础上的相互作用，将强调探测病毒生命周期的各个方面。这些方法将被整合，并将设计一个迭代过程，以提取最佳信息和完善实验方向。在高度协作的环境中，采用晶体学，冷冻电子显微镜，生物化学，分子生物学和遗传学的单个项目的协同作用是我们提案的关键优势。这种结构-功能方法应该为理解这些病毒的生命周期和发病机制提供丰富的信息，这在疫苗和抗病毒药物的设计中将是非常宝贵的，并为理解病毒发病机制提供知识基础。

Program Director/Principal Investigator (Last, First, Middle): Kuhn, Richard J. 2 P01 AI055672-06A1 ABSTRACT The current understanding of alphavirus and flavivirus life cycles at the molecular level is incomplete because we lack a structural foundation for the viral and host proteins involved in RNA replication, genome packaging and particle assembly. Viruses within these two groups pose significant risks to large segments of the population and methods for controlling infection and disease are few. Therefore, we will continue our systematic collaborative investigation that will provide a comparative approach to the structure and function of the virally encoded proteins representing pathogens within these two groups. We will shift our emphasis to encompass not only viral proteins but also host proteins and the appropriate complexes between host and pathogen. The tools proposed for studying the structure and function of these viral proteins are varied and multidisciplinary. We previously placed a heavy emphasis on X-ray crystallography of individual viral proteins and intact viruses (Michael Rossmann, Jan.et Smith). In addition to our emphasis on high-resolution structures from crystallography, a key role is played by Tim Baker, Wen Jiang, and Michael Rossmann in the cryoelectron microscopy study of viruses, complexes of receptors and antibodies, and intermediates in their assembly/disassembly. Electron tomography and immuno-electron microscopy (Richard Kuhn, Wen Jiang, Michael Rossmann) will also be employed as a means to image subcellular structures induced and/or utilized during virus infection. Functional biochemical and genetic analyses (Richard Kuhn, Janet Smith) will complement and support the structural studies. Site-directed mutagenesis and protein interaction approaches to explore interactions found, or hypothesized, on the basis of structure determination will be emphasized to probe various aspects of the virus life cycle. These approaches will be integrated and an iterative process will be designed for extracting optimal information and refining experimental directions. The synergy of individual projects, which employ crystallography, cryo-electron microscopy, biochemistry, molecular biology, and genetics, in a highly collaborative environment is a critical strength of our proposal. This structural-functional approach should provide a wealth of information for understanding the life cycle and pathogenesis of these viruses that will be invaluable in the design of vaccines and antivirals, as well as providing a knowledge base for understanding virus pathogenesis.