Structural Studies of Alphavirus Membrane Fusion In Situ

描述(申请人提供)：甲型病毒是囊膜病毒，外加Togaviridae家族的链状RNA病毒，是人类和动物的重要病原体。这些病毒通过受体介导的内吞作用途径入侵宿主细胞。内体中的酸性环境诱导病毒和内体膜的融合，允许病毒基因组进入受感染细胞的细胞质。合并水化膜的能量成本被病毒E1糖蛋白(一种II类融合蛋白)的构象变化和寡聚体重排所克服。虽然已经确定了融合前和融合后构象中E1胞外区的晶体结构，但对融合蛋白在膜融合过程中中间组织的结构细节了解很少。阻碍深入结构研究的主要障碍是低pH条件下病毒颗粒的聚集和异质性，以及不均匀的靶膜引入的额外的异质性。这项拟议的研究采用了几种新的程序来克服这些困难，并在酸性pH下生产与脂膜复合的合格的甲型病毒样本。低温电子显微镜和三维图像重建技术将被用来确定纳米分辨率的病毒-膜复合体的结构。将E1蛋白的晶体结构拟合到重建图中，将导致E1在酸化后插入靶膜时的构象、寡聚和组织的发现。这项拟议研究的长期目标是概述与膜重塑和融合相关的病毒蛋白构象变化的详细步骤。从这项研究中获得的经验可以推广并应用于涉及其他小包膜病毒的类似结构研究，如黄病毒。在这项研究中获得的知识也将有助于抗病毒策略的发展。公共卫生相关性：流感病毒、西尼罗河病毒和辛德比斯病毒等被包膜病毒感染宿主细胞的关键步骤是病毒被膜与宿主细胞膜的融合。本研究旨在阐明Sindbis病毒融合蛋白驱动这一过程的结构细节，并为研究包膜病毒膜融合的结构提供一种通用的方法。从这些研究中获得的知识为抗病毒试剂提供了潜在的靶点。

DESCRIPTION (provided by applicant): The Alphaviruses are enveloped, plus strand RNA viruses within the Togaviridae family, and are important human and animal pathogens. These viruses invade the host cells through the receptor-mediated endocytosis pathway. The acidic environment in the endosome induces fusion of the viral and endosomal membranes, allowing delivery of the viral genome into the cytoplasm of the infected cell. The energy cost for merging the hydrated membranes is overcome by the conformational changes and oligomeric rearrangements of the viral E1 glycoproteins, a class-II fusion protein. Although the crystal structures of the E1 ecto-domain in pre- and post-fusion conformations were determined, the structural details of the intermediate organizations of the fusion protein during the course of membrane fusion are poorly understood. Major obstacles that have impeded vigorous structural studies are the aggregation and heterogeneity of virus particles at low-pH, and additional heterogeneity introduced by nonuniform target membranes. The proposed research employs several novel procedures to overcome such difficulties and produce eligible samples of Alphavirus in complex with lipid membranes at acidic pH. Cryo-electron microscopy and three-dimensional image reconstruction techniques will be used to determine the structure of the virus-membrane complex at nanometer resolution. Fitting the crystal structures of the E1 protein into the reconstruction map will lead to discovery of the conformation, oligomerization and organization of E1 when it inserts into a target membrane upon acidification. The long-term objective of the proposed research is to outline the detailed steps of viral protein conformational changes correlating with membrane remodeling and fusion. The experience obtained from this study can be generalized and applied to similar structural investigations that involve other small-enveloped viruses, such as Flaviviruses. Knowledge gained in this study will also be beneficial for the development of antiviral strategies. PUBLIC HEALTH RELEVANCE: A key step in the infection of enveloped viruses, such as influenza virus, West Nile virus and Sindbis virus, into the host cell, is fusion of the virus envelope with the host cell membrane. This research aims to illustrate the structural details of this process driven by the Sindbis viral fusion proteins, and provides a general approach for structural investigation of membrane fusion carried out by the enveloped viruses. Knowledge gained from such studies provides potential targets for anti-viral reagents.