Rabies virus glycoprotein structure determination and receptor binding interactions

项目总结/摘要 狂犬病病毒每年造成5万多人死亡，如果不治疗，几乎100%致命。狂犬病 在美国和世界各地的动物自然水库种群中是地方性的，并且宿主- 适应狂犬病病毒株存在于许多这些水库人口，包括蝙蝠，臭鼬， 浣熊狂犬病病毒糖蛋白（RabvG）负责与宿主细胞上的受体结合，并且是狂犬病病毒的主要抗原。 抗体反应的主要目标和疫苗的主要成分。然而，它的结构并没有 确定，很少有人知道宿主适应突变RabvG如何影响受体结合， 传染性为了设计改进的狂犬病病毒抗病毒疗法和疫苗， 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 与受体和抗体的相互作用。本项目的目的是（1）定量RabvG 及其三种细胞受体;（2）表征抗体与RabvG的结合，并确定 抗体结合对RabvG/受体复合物的影响;（3）表达和纯化RabvG，单独或复合 与受体或抗体一起，作为构象均匀的群体，通过冷冻进行结构测定， 电子显微镜（cryo-EM）或断层摄影术。 我们将进行生物层干涉实验，以量化RabvG与 及其三种细胞受体（神经细胞粘附分子（NCAM 1）、p75神经营养因子受体 （p75 NTR）和烟碱乙酰胆碱受体（nAChR））。我们将比较RabvG的结合亲和力， 从不同的宿主适应性狂犬病病毒株到来自相应和不同宿主物种的受体，和 然后测量用RabvG假型化的水泡性口炎病毒（VSV）对表达 不同的宿主物种受体，以确定受体结合亲和力是否与感染性相关。 我们还将使用生物层干涉测量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/受体复合物以中和病毒。最后，这些受体和抗体结合 研究将为RabvG的工程和优化提供信息，以通过cryo-EM进行结构测定。 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， 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， 预防治疗。解决RabvG结构将提供模板，以更好地了解病毒进入， 抗体中和，为其他威胁人类健康的狂犬病毒提供模型，并有助于进一步研究 开发广泛保护性疫苗。

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.