Structural Studies of Noroviruses

项目概要：项目3 项目3的主要目标是提供一个详细的结构和机制的理解， 诺如病毒（NoV）的进化和复制，以帮助设计和开发抗病毒策略。人类 NoV是流行性急性胃肠炎的主要原因。对这些病毒的敏感性由以下因素决定： 组织血型抗原（HBGAs）的遗传控制表达，这对NoV也至关重要 附着在宿主细胞上由于衣壳蛋白VP1的P结构域的序列变化， 具有HBGA结合位点，这些病毒在HBGA特异性方面表现出菌株依赖性变异性 提出了一个迷人的案例研究，在如何基因型变异允许利用多态性 宿主人群中HBGAs的性质，以对抗群体免疫并引起流行病。最近的研究 显示阻断HBGA结合的人抗体与保护免受NovV相关疾病相关 （项目1）。一些研究表明抗原性和HBGA特异性之间存在相互作用 in driving驱动the evolution进化of NoV新能源汽车. AIM 1的目标是为这种相互作用提供结构基础， 使用目前可用的方法，在结构上表征HBGA阻断抗体如何与NoV毒株相互作用 人单克隆抗体和项目1中新开发的抗体，使用X射线晶体学， 冷冻电镜技术虽然AIM 1的重点是病毒与宿主的相互作用，但我们以下的重点 两个AIM是两个关键的病毒编码蛋白，调节病毒复制，这两个都是潜在的 小分子药物发现的目标。AIM2主要研究病毒蛋白酶，它对多聚蛋白的形成至关重要 处理.基于我们和其他人最近令人兴奋的发现，即NoV蛋白酶与病毒RNA结合，我们 已经假设它在基因组复制中起着迄今为止未被描述的作用。在AIM的第一部分中 我们的目标是确定基因组依赖性底物相互作用和抑制的结构基础 为了提供一个合理的框架，设计和优化抑制剂的合作， 项目1。在该目标的第二部分中，我们的目标是从结构上表征NoV蛋白酶与 病毒RNA，并与项目2合作，探讨这种相互作用在病毒复制中的作用。AIM 3 将集中在诺瓦克病毒NTR，p41，一个高度保守的蛋白质家族的成员编码的一个蛋白质， 多种（+）RNA病毒，通过重塑细胞膜在病毒复制中起关键作用 进入受感染细胞的囊泡区室。在目前的资助期间，我们提供了第一个结构性的 从我们对p41的研究中鉴定了这种蛋白质。这一目标的目标是采取下一步行动， 使用晶体学、冷冻电镜和基于细胞的 技术，并将我们的观察结果与病毒复制过程中这种蛋白质的功能方面联系起来 与项目2合作。我们希望我们提出的研究将提供新的结构和机制， 这将对免疫学，翻译和复制相关方面产生重大影响， 人NoV。

PROJECT SUMMARY: Project 3 The major goal of Project 3 is to provide a detailed structural and mechanistic understanding of human norovirus (NoV) evolution and replication to aid in the design and development antiviral strategies. Human NoVs are the leading cause of epidemic acute gastroenteritis. Susceptibility to these viruses is determined by genetically controlled expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. As a result of sequence changes in the P domain of the capsid protein VP1 that harbors the HBGA binding site, these viruses show strain-dependent variability in HBGA specificity presenting a fascinating case study in how genotypic variations allow for exploitation of the polymorphic nature of HBGAs in host populations to counter herd immunity and cause epidemics. Recent studies have shown human antibodies that block HBGA binding correlate with protection from NoV-associated illness (Project 1). Several studies have suggested a correlated interplay between antigenicity and HBGA specificity in driving the evolution of NoVs. The goal of AIM 1 is to provide the structural basis for such interplay by structurally characterizing how HBGA-blocking antibodies interact with NoV strains using currently available human monoclonal antibodies and those newly developed in Project 1, using X-ray crystallography and cryo-EM techniques. While the emphasis in AIM 1 is on virus-host interactions, the emphasis in our following two AIMs is on two key virus encoded proteins that regulate virus replication, both of which are potential targets for small molecule drug discovery. AIM 2 focuses on the viral protease that is critical for polyprotein processing. Based on a recent exciting finding by us and others that NoV protease binds to viral RNA, we have hypothesized that it plays a hitherto uncharacterized role in genome replication. In the first part of AIM 2, our goal is to determine the structural basis of genogroup-dependent substrate interactions and inhibition in order to provide a rational framework for the design and optimization of inhibitors in collaboration with Project 1. In the second part of this aim, our goal is to structurally characterize NoV protease interaction with viral RNA, and in collaboration with Project 2, probe into the role of this interaction in viral replication. AIM 3 will focus on Norwalk virus NTPase, p41, a member of a highly conserved family of proteins encoded by a wide variety of (+)RNA viruses that plays a critical role in virus replication by remodeling cellular membranes into vesicular compartments in infected cells. During the current grant period, we provided the first structural characterization of such a protein from our studies on p41. The goal of this aim is to take the next step of addressing mechanism-related questions using a combination of crystallography, cryo-EM and cell-based techniques and to correlate our observations to the functional aspects of this protein during virus replication in collaboration with Project 2. We expect our proposed studies will provide novel structural and mechanistic insight that will have significant impact on immunological, translational, and replication-related aspects of human NoVs.