Structural Studies of Noroviruses

项目总结：项目3 项目3的主要目标是提供对人类的详细结构和机械理解 诺沃克病毒(NOV)的进化和复制，以帮助设计和开发抗病毒策略。人类 新城疫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(HBGA)的基因控制表达，这也是NOV的关键 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， 含有HBGA结合位点，这些病毒在HBGA特异性上表现出依赖于菌株的变异性 提供了一个有趣的案例研究，说明了基因变异如何允许利用多态 HBGA在宿主人群中的性质，以对抗群体免疫和引起流行病。最近的研究表明 显示阻断HBGA结合的人类抗体与预防NOV相关疾病相关 (项目1)。多项研究表明，抗原性和HBGA特异性之间存在相互作用 推动新能源汽车的发展。目标1的目标是通过以下方式为这种相互作用提供结构基础 目前可用的HBGA阻断抗体与NOV毒株相互作用的结构特征 人类单抗和项目1中新开发的单抗，使用X射线结晶学和 低温电磁技术。虽然AIM 1的重点是病毒与宿主的相互作用，但我们下面的重点是 两个目标是针对控制病毒复制的两个关键病毒编码蛋白，这两个蛋白都是潜在的 小分子药物发现的靶点。目标2重点介绍对多蛋白起关键作用的病毒蛋白酶 正在处理。基于我们和其他人最近的一项令人兴奋的发现，即Nov蛋白酶与病毒RNA结合，我们 都假设它在基因组复制中扮演着迄今未被描述的角色。在AIM的第一部分 2、我们的目标是确定依赖于基因组的底物相互作用和抑制的结构基础 为了给缓蚀剂的设计和优化提供一个合理的框架 项目1。在这个目标的第二部分，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。目标3 将集中在诺沃克病毒NTPase，p41，一个高度保守的蛋白质家族的成员，由一个 多种(+)RNA病毒，通过重塑细胞膜在病毒复制中发挥关键作用 进入受感染细胞的囊泡隔间。在目前的赠款期间，我们提供了第一个结构 从我们对p41的研究中对这种蛋白的特性进行了研究。这一目标的目标是采取下一步 使用结晶学、低温EM和细胞学相结合的方法解决机制相关问题 技术，并将我们的观察结果与病毒复制过程中该蛋白的功能方面相关联 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械 洞察力将对免疫、翻译和复制相关方面产生重大影响 人类新人。

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.