Structure-function studies of alpha- and flaviviruses

目前在分子水平上对甲型病毒和黄病毒生命周期的了解是有限的，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。这两个群体中的病毒对很大一部分人口构成重大风险，控制感染和疾病的方法很少。因此，我们建议继续我们的系统合作调查，以提供一种比较的方法来 代表这两组病原体光谱的病毒编码蛋白的结构和功能。在这个提案中，我们将把重点转移到不仅包括病毒蛋白，而且还包括宿主蛋白和适当的BEH/VEN宿主和病原体复合体。为研究这些病毒蛋白的结构和功能而提出的工具是多样的和多学科的。我们以前非常重视单个病毒蛋白和完整病毒的X射线结晶学(Michael Rossmann，Janet Smith)。除了我们强调来自结晶学的高分辨率结构外，Tim Baker、温江和Michael Rossmann在病毒、受体和抗体的复合体以及它们组装/拆卸的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成像在病毒感染期间诱导和/或使用的亚细胞结构的手段。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。将重点介绍定点突变和蛋白质相互作用的方法，以探索在结构确定的基础上发现的或假设的相互作用，以探索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，使用结晶学、低温电子显微镜、生物化学、分子生物学和遗传学的各个项目的协同作用是我们提案的关键优势。这种结构-功能方法应该为了解这些病毒的生命周期和致病机理提供丰富的信息，这些信息对疫苗和抗病毒药物的设计将是非常宝贵的，并在与这些病毒出现或重新出现有关的紧急情况下提供知识库。

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.