Rabies virus glycoprotein structure determination and receptor binding interactions

项目摘要/摘要 狂犬病病毒每年导致5万多人死亡，如果不治疗，几乎是100%致命的。狂犬病 在美国和世界各地的自然水库动物种群中是地方性的，宿主- 适应狂犬病病毒的毒株存在于许多这样的水库种群中，包括蝙蝠、臭鼬和 浣熊。狂犬病病毒糖蛋白(RabvG)负责与宿主细胞上的受体结合，是 抗体反应的主要靶点和疫苗的主要成分。然而，它的结构并不是 已确定且鲜为人知的是，宿主适应性RabvG突变如何影响受体结合和 传染性。为了设计改进的狂犬病病毒抗病毒疗法和疫苗，并更好地了解 狂犬病病毒感染时，有必要确定RabvG的结构并彻底检查其结合情况 与受体和抗体的相互作用。本项目的目的是(1)量化RabvG之间的结合亲和力 以及它的三种细胞受体；(2)鉴定与RabvG结合的抗体并确定其作用 抗体与RabvG/受体复合体的结合；(3)表达和纯化RabvG 与受体或抗体，作为构象一致的群体，用于通过冷冻- 电子显微镜(冷冻-EM)或断层扫描。 我们将进行生物层干涉实验来量化RabvG之间的结合亲和力 以及它的三种细胞受体(神经细胞黏附分子(NCAM1)、p75神经营养素受体 (P75NTR)和烟碱型乙酰胆碱受体(NAChR))。我们将比较RabvG的结合亲和力 从不同宿主适应的狂犬病病毒株到相应和不同宿主物种的受体，以及 用RabvG检测水疱性口炎病毒(VSV)对表达细胞的感染力 为了确定受体结合亲和力是否与感染性相关，对不同寄主物种的受体进行研究。 我们还将使用生物层干涉法来确定相对抗体亲和力，以及抗体是否可以破坏前 形成RabvG/受体复合体，中和病毒。最后，这两种受体和抗体的结合 研究将为RabvG的工程和优化提供信息，以便用低温EM确定结构。 这些实验将产生关于病毒/受体/抗体结合相互作用的新信息，这些信息可以 用于预测狂犬病病毒溢出事件和宿主范围跳跃，以及提高免疫力 预防性治疗。解决RabvG结构将提供模板，以更好地了解病毒进入和 抗体中和，为威胁人类健康的其他溶血病毒提供一个模型，并有助于进一步 开发具有广泛保护性的疫苗。

Project Summary/Abstract Rabies virus causes more than 50,000 deaths every year and is nearly 100% lethal if untreated. Rabies is endemic in natural reservoir populations of animals in the United States and throughout the world, and host- adapted rabies virus strains exist for many of these reservoir populations, including bats, skunks, and raccoons. The rabies virus glycoprotein (RabvG) is responsible for binding to receptors on host cells and is the major target of the antibody response and a major component of vaccines. Its structure, however, has not been determined and little is known about how host-adaptation mutations to RabvG affect receptor binding and infectivity. In order to design improved antiviral therapies and vaccines for rabies virus and to better understand rabies virus infection, it is necessary to determine the structure of RabvG and to thoroughly examine its binding interactions with receptors and antibodies. This project aims (1) to quantify the binding affinity between RabvG and its three cellular receptors; (2) to characterize antibody binding to RabvG and to determine what effects antibody binding has on RabvG/receptor complexes; and (3) to express and purify RabvG, alone or in complex with receptor or antibody, as a conformationally uniform population for structural determination via cryo- electron microscopy (cryo-EM) or tomography. We will conduct bio-layer interferometry experiments to quantify the binding affinity between RabvG and its three cellular receptors (the neural cell adhesion molecule (NCAM1), the p75 neurotrophin receptor (p75NTR), and the nicotinic acetylcholine receptor (nAChR)). We will compare the binding affinity of RabvG from different host-adapted rabies virus strains to receptors from corresponding and different host species, and then measure the infectivity of vesicular stomatitis virus (VSV) pseudotyped with RabvG on cells expressing the different host species receptors in order to determine if receptor binding affinity correlates with infectivity. We will also use bio-layer interferometry to determine relative antibody affinity and if antibodies can disrupt pre- formed RabvG/receptor complexes to neutralize virus. Finally, both of these receptor and antibody binding studies will inform engineering and optimization of RabvG for structural determination by cryo-EM. These experiments will yield new information about virus/receptor/antibody binding interactions that can be used to predict rabies virus spillover events and host range jumps as well as to improve immuno- prophylaxis treatments. Solving the RabvG structure will provide templates to better understand viral entry and antibody neutralization, yield a model for other lyssaviruses that threaten human health, and aid in further development of broadly protective vaccines.