Cryo-Electron and Biochemical Analysis of Native Paramyxovirus Fusion Complexes

描述（由申请方提供）：蛋白质介导的膜融合对于维持真核细胞组织和主要人类病毒的繁殖至关重要。副粘病毒家族的许多临床相关成员依赖于两种包膜糖蛋白（附着蛋白和融合蛋白）的协同作用，将其包膜与靶细胞质膜融合，以进入细胞。然而，尽管它们的临床重要性，基本的机械原理，管理的组织和功能的天然副粘病毒融合复合物不了解。为了阐明这些原则的总体目标，本项目将在三个基本问题中探讨这个问题：感染性颗粒表面上显示的天然亚稳态构象中的包膜糖蛋白异源寡聚体复合物的空间组织是什么？受体结合如何影响附着蛋白的结构？是什么分子机制将受体结合与融合蛋白重折叠成生物学稳定的融合后构象联系起来？ 随着不同副粘病毒糖蛋白的分离胞外结构域的晶体结构在手，拟议的研究将集中在麻疹病毒包膜糖蛋白，以解决这些问题，在一个全面的，跨学科的方法，接口创新的成像，生物化学，功能和计算实验策略。冷冻电子断层扫描结合包膜糖蛋白工程将阐明单独和用可溶性受体处理后病毒颗粒表面上显示的水合融合复合物的整体空间组织（目的1）。天然凝胶电泳、H寡聚体稳定化和去稳定化修饰以及H双分子互补将通过评估受体结合对附着蛋白组织的影响以及表征启动融合蛋白重折叠的信号的分子性质来提取功能信息（目的2）。分子建模引导的诱变，生物化学接触域映射，肽结合和质谱将交叉检查，扩大和功能特性的候选人在试点研究中发现的分子间接触，导致在任一糖蛋白中的离散微结构域的识别线的异源寡聚体界面和控制天然副粘病毒融合复合物的结构完整性（目标3）。 公共卫生相关性：阐明病毒如何在分子水平上感染人类细胞不仅对于更好地理解病毒已经开发的基本细胞进入机制至关重要，而且对于设计可以阻断病毒感染的新药也至关重要。副粘病毒是一组引起主要人类疾病的病毒，包括麻疹、腮腺炎和流感样疾病，以及几种重要的动物疾病。该项目将确定负责病毒进入人类细胞的副粘病毒表面蛋白质的排列，并精确定义这些蛋白质如何共同介导感染的机制。尽管这些问题已被深入调查，但尚未得到解答。从这项研究中获得的见解将通过未来开发创新的抗病毒药物来改善公共卫生，这些药物可以有效地阻止这组重要病毒进入过程的重要步骤。

DESCRIPTION (provided by applicant): Protein-mediated membrane fusion is essential for maintaining eukaryotic cell organization and propagation of major human viruses. Many clinically relevant members of the paramyxovirus family rely on the concerted action of two envelope glycoproteins, the attachment and fusion protein, to fuse their envelope with the target cell plasma membrane for cell entry. However, despite their clinical importance, fundamental mechanistic principles that govern the organization and function of native paramyxovirus fusion complexes are not understood. Towards the overarching goal of elucidating these principles, this project will pursue the problem in three basic questions: What is the spatial organization of the envelope glycoprotein hetero-oligomer complexes in the native, metastable conformation displayed on the surface of infectious particles? How does receptor binding affect attachment protein organization? What is the molecular mechanism that links receptor binding with fusion protein refolding into the thermodynamically stable postfusion conformation? With crystal structures of isolated ectodomains of different paramyxovirus glycoproteins at hand, the proposed studies will focus on measles virus envelope glycoproteins to address these questions in a comprehensive, interdisciplinary approach that interfaces innovative imaging, biochemical, functional, and computational experimental strategies. Cryo-electron tomography combined with envelope glycoprotein engineering will elucidate the overall spatial organization of hydrated fusion complexes displayed on the surface of viral particles, alone and after treatment with soluble receptor (aim 1). Native gel electrophoresis, H oligomer stabilizing and destabilizing modifications, and H bimolecular complementation will extract functional information by assessing the effect of receptor binding on attachment protein organization and characterizing the molecular nature of the signal that initiates fusion protein refolding (aim 2). Molecular modeling-guided mutagenesis, biochemical contact domain mapping, peptide binding and mass spectrometry will cross-examine, expand and functionally characterize candidate intermolecular contacts found in pilot studies, resulting in the identification of discrete microdomains in either glycoprotein that line the hetero-oligomer interface and control the structural integrity of a native paramyxovirus fusion complex (aim 3). PUBLIC HEALTH RELEVANCE: Elucidating how viruses infect human cells at a molecular level is crucial not only for better understanding the basic cell entry mechanisms that viruses have developed, but also for the design of new drugs that can block viral infection. Paramyxoviruses are a group of viruses that cause major human diseases, including measles, mumps, and flu-like illnesses, and several important animal diseases. This project will determine the arrangement of proteins on the paramyxovirus surface that are responsible for virus entry into human cells, and define precisely the mechanism of how these proteins work together to mediate infection. Although much-investigated, these questions have yet to be answered. Insight gained from this study will improve public health by enabling the future development of innovative antiviral drugs that efficiently block essential steps of the entry process of this important group of viruses.