Structure-function studies of alpha- and flaviviruses

目前在分子水平上对甲病毒和黄病毒生命周期的理解是有限的，因为我们缺乏病毒和宿主蛋白的结构基础，特别是那些参与RNA复制，基因组包装和颗粒组装的蛋白。这两类病毒对大部分人群构成重大风险，控制感染和疾病的方法很少。因此，我们建议继续我们的系统合作调查，这将提供一个比较的方法， 病毒编码的蛋白质的结构和功能代表了这两组病原体的谱。在这个建议中，我们将把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质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，Janet 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。电子断层扫描和免疫电子显微镜也将被用作对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 （理查德·库恩，珍妮特·史密斯）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-功能的方法应该为理解这些病毒的生命周期和发病机制提供了丰富的信息，这在疫苗和抗病毒药物的设计中将是非常宝贵的，并且如果出现与这些病毒的出现或重新出现有关的紧急情况，也可以提供知识基础。

The current understanding of alphavirus and flavivirus life cycles at the molecular level is limited because we lack a structural foundation for the viral and host proteins, especially those involved in RNA replication, genome packaging and particle assembly. Viruses within these two groups pose significant risks to large segments ofthe population and methods for controlling infection and disease are few. Therefore, we propose to continue our systematic collaborative investigation that will provide a comparative approach to the structure and function of the virally encoded proteins representing the spectrum of pathogens within these two groups. In this proposal, we will shift our emphasis to encompass not only viral proteins but also host proteins and the appropriate complexes beh/veen host and pathogen. The tools proposed for studying the structure and function of these viral proteins are varied and multi-disciplinary. We previously placed a heavy emphasis on X-ray crystallography of individual viral proteins and intact viruses (Michael Rossmann, Janet 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 cryo-electron microscopy study of viruses, complexes of receptors and antibodies, and intermediates In their assembly/disassembly. Electron tomography and immuno-electron microscopy will also be employed as a means to image sub-cellular 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 should there be an emergency related to the emergence or reemergence of these viruses.