Structural Studies of Noroviruses

项目摘要--项目3 项目3的主要目标是提供对人类各个方面的详细结构理解 诺沃克病毒(HuNoV)生物学协同补充项目1和2中的研究，并帮助 设计和开发抗病毒策略。新城疫病毒是引起流行性急性胃肠炎的主要原因。 这些病毒表现出巨大的基因多样性，并在此基础上被划分为几个基因组组 和基因分型。组织血型的基因控制表达决定了对人类新城疫病毒的易感性 抗原(HBGA)，这也是它们与宿主细胞附着的关键。在之前的授权期内， 广泛的结构研究，我们已经表明序列变化如何改变HBGA结合特异性和 它们如何影响抗原性格局以推动这些病毒的进化。项目1中的研究 目前的授权期，通过识别几个阻断人类单抗的HBGA，进一步放大了这一概念 具有意想不到的广度和中和活性的各种同种类型的抗体(MAbs)使用 人类肠道类病毒(HIE)系统是在项目2中开发的。在此期间，我们在项目3中的研究有 而GI.1特异性单抗的阻断机制是通过直接阻断HBGA结合部位 GI.1 HuNov，与交叉反应的GII特异性mAb，阻断/中和机制不同 可能是独一无二的。目标1的目的是加深我们对潜在的不同机制的理解 用X射线结晶学、冷冻-EM等技术阻断/中和人单抗对HuNov的作用 补充生物物理技术。我们以下两个目标的重点是非结构蛋白。 为设计和优化新型病毒提供了一个结构框架 通过高通量筛选程序发现的抗病毒化合物(项目1和 2)。AIM 2旨在提供对GII.4蛋白水解酶及其独特功能的结构理解 与底物相互作用，新发现的小分子如何抑制GI和GII.4HuNoV蛋白酶 和GII-4聚合酶，以及在筛选过程中发现一些小分子的机制是什么 与VPG类似，可增强聚合酶活性。AIM 3的研究重点是G1.1HuNoV编码的p41蛋白。 P41具有膜锚定结构域、NTPase和解旋酶活性，在病毒中起着关键作用 复制。在之前的授权期中，我们确定了p41的第一个无膜晶体结构。 锚定域。尽管它提供了对NTPase活动的结构性洞察，但它不足以提供 对潜在的膜相关解旋酶，RNA重塑/RNA的完整机制的理解 P41的伴侣活性。基于令人兴奋的初步数据，AIM 3的研究将集中于确定 用低温电子显微镜和X射线结晶学研究可溶纳米盘重组全长p41的结构 方法：研究方法。我们希望我们提出的研究将提供新的结构性和机械性的见解，将具有 对HuNov的免疫学、翻译和复制相关方面产生重大影响。

PROJECT SUMMARY –Project 3 The major goal of Project 3 is to provide a detailed structural understanding of various aspects human norovirus (HuNoV) biology to synergistically complement the studies in Projects 1 and 2, and aid in the design and development of antiviral strategies. HuNoVs are the leading cause of epidemic acute gastroenteritis. These viruses show enormous genotypic diversity and based on this they are classified into several genogroups and genotypes. Susceptibility to HuNoVs is determined by genetically controlled expression of histo-blood group antigen (HBGA), which is also critical for their attachment to host cells. In the previous grant periods, from extensive structural studies, we have shown how the sequence changes alter the HBGA binding specificity and how they impact the antigenic landscape to drive the evolution of these viruses. Studies in Projects 1 during the current grant period, have further amplified this notion by identifying several HBGA blocking human monoclonal antibodies (mAbs) of various isotypes with an unexpected degree of breadth and neutralization activity using the human intestinal enteroid (HIE) system developed in Project 2. Our studies in Project 3 during this period have shown while the blockade mechanism of a GI.1-specific mAb is by directly blocking the HBGA binding site in GI.1 HuNoV, with a cross-reactive GII-specific mAb, the blockade/neutralization mechanism is different and potentially unique. The goal of AIM 1 is to further our understanding of potentially varying mechanisms of blockade/neutralization of HuNoVs by human mAbs using X-ray crystallography, cryo-EM, and other complementary biophysical techniques. The emphasis in our following two AIMs is on the non-structural proteins of HuNoV that regulate virus replication to provide a structural framework for design and optimization of novel antiviral compounds that are being discovered through high-throughput screening procedures (Projects 1 and 2). AIM 2 is designed to provide a structural understanding of how GII.4 protease with its unique features interacts with the substrates, how the newly discovered small molecules inhibit GI and GII.4 HuNoV proteases and GII.4 polymerase, and what is the mechanism by which some small molecules discovered during screening similar to VPg enhance polymerase activity. The focus of AIM 3 is on the p41 protein encoded by G1.1 HuNoV. p41, with its membrane-anchoring domain, NTPase and helicase activities, plays a critical role in virus replication. In the previous grant period, we determined the first crystal structure of p41 lacking the membrane anchoring domain. Although it provided structural insight into the NTPase activity, it was not sufficient to provide a complete mechanistic understanding of the potentially membrane-associated helicase, RNA remodeling/RNA chaperone activity of p41. Based on the exciting preliminary data, studies in AIM 3 will focus on determining the structure of the soluble and nanodisc-reconstituted full-length p41 using cryo-EM and X-ray crystallographic methods. We expect our proposed studies will provide novel structural and mechanistic insight that will have a significant impact on immunological, translational, and replication-related aspects of HuNoVs.