Structure and Assembly of Viruses

病毒的结构和组装

• 批准号: 10304898
• 负责人: STEPHEN COPLAN HARRISON
• 金额: $ 41.2万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-06-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10304898
• 关键词: Capsid; Cell membrane; Cell surface; Cell-Matrix Junction; Cells; Chimeric Proteins; Collaborations; Coupled; Cryoelectron Microscopy; Crystallography; Cytosol; Data; Dengue Virus; Detection; Dimensions; Double Stranded RNA Virus; E protein; Electron Microscopy; Electrons; Elements; Event; Flavivirus; Fluorescence Microscopy; Foundations; Goals; Grant; In Vitro; Infection; Ions; Label; Light; Lipid Bilayers; Liposomes; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Mutation; Outcome Study; Pathogenesis; Pathway interactions; Penetration; Perforation; Process; Property; Proteins; RNA Viruses; RNA chemical synthesis; Recombinant Proteins; Recombinants; Resolution; Rhesus; Rotavirus; Site; Structure; System; Testing; Time; Tropism; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus-like particle; West Nile virus; Work; cell type; design; experimental study; human pathogen; imaging study; live cell imaging; movie; novel strategies; particle; recombinant virus; reconstitution; single molecule; uptake; virus envelope

Summary The structure of a virus particle must conform to the mechanism by which it enters and infects a cell. We seek to relate structures of virus particles and viral surface proteins to mechanisms of the steps in cell attachment, uptake and penetration that initiate a productive infection. The proposed experiments take advantage of recent advances in electron cryomicroscopy (cryo-EM) and live-cell imaging to define these mechanisms for both non- enveloped and enveloped viruses, taking rotaviruses and flaviviruses (West Nile and dengue viruses, in particular) as specific examples. Previous live-cell imaging studies defined the cellular entry pathway for rhesus rotavirus (RRV). We can now relate structures we have determined for distinct conformations of the spike-like viral protein 4 (VP4), which mediates cell entry, to stages of plasma-membrane remodeling in the entry pathway. We will extend on-going analyses by cryo-EM of VP4 conformations at the membrane interface, both on cells and in an in vitro-reconstituted system for early steps, to determine how RRV deforms and then disrupts a membrane bilayer. A mechanism for RRV membrane perforation, suggested by preliminary data, may help unify observations made with several other RNA viruses. Proposed experiments will connect high- resolution structures with direct analyses (by electron cryotomography and potentially by high-resolution template matching) of VP4 conformations during cell entry and with sequences of events in time (by lattice- light-sheet microscopy). For the flavivirus fusion protein (the "E protein"), completing a structure for the postfusion conformation of the intact protein will enable design of experiments to trap fusion intermediates and thus to define the fusion mechanism more completely than hitherto possible. The intended outcomes of the studies, for both rotaviruses and flaviviruses, are structurally and mechanistically accurate "molecular movies" of viral entry.

概括 病毒颗粒的结构必须符合其进入并感染细胞的机制。我们寻找 将病毒颗粒和病毒表面蛋白的结构与细胞附着的步骤的机制联系起来， 引发生产感染的吸收和穿透性。提出的实验利用了最近的优势 电子冷冻显微镜（Cryo-EM）和活细胞成像的进步，以定义两种非 - 包裹和包裹的病毒，服用轮状病毒和黄病毒（西尼罗河和登革热病毒， 特别）作为特定示例。以前的活细胞成像研究定义了细胞进入途径 轮状病毒（RRV）。现在，我们可以将我们确定的结构联系起来 尖峰样病毒蛋白4（VP4）介导细胞进入的尖峰状病毒蛋白4 入口途径。我们将通过膜界面处的VP4构象的冷冻EM扩展进行进行的分析， 在细胞和体外重新建立的系统上，以进行早期步骤，以确定RRV如何变形，然后 破坏膜双层。 RRV膜穿孔的机制，由初步数据提出， 可能有助于统一使用其他几种RNA病毒进行的观察结果。建议的实验将连接高 具有直接分析的分辨率结构（通过电子冷冻理学，并有可能通过高分辨率 VP4构象的模板匹配）在细胞进入过程中和及时序列（通过晶格 - 灯表显微镜）。对于黄病毒融合蛋白（“ E蛋白”），完成了 完整蛋白质的灌注后构象将使实验设计以捕获融合中间体和 因此，比迄今为止可能更完全定义融合机制。预期的结果 对于轮状病毒和黄病毒，研究在结构和机械上都是准确的“分子电影” 病毒进入。