Interplay of protein and membrane intermediates during influenza virus fusion

描述(申请人提供)：蛋白介导膜融合是一种基本的生物过程，它位于包膜病毒感染、突触信号传递、细胞内小泡运输、配子受精和细胞-细胞融合的核心。尽管进行了深入的研究，但目前我们对融合蛋白机制如何操纵类脂膜以诱导它们融合的机制了解有限。被包裹的病毒使用特殊的蛋白质机制将它们的膜与宿主细胞膜融合，并传递它们的遗传物质以供复制。在流感病毒中，三聚体血凝素(HA)糖蛋白尖峰负责宿主细胞的附着和膜融合。虽然已经表征了融合机制的构象和部分的子集的结构，但驱动融合过程的状态已被证明是难以确定经典结构的。此外，融合过程中膜变形的性质在很大程度上还无法确定。从本质上讲，我们缺乏对膜融合和病毒进入的机械理解。这项建议侧重于确定血凝素和膜在融合过程中的结构和功能相互作用。将采用荧光光谱、电子冷冻断层扫描(ECT)、小角X射线散射(SAXS)和三维形状重建以及氢-氚交换-质谱分析(HD-MS)相结合的方法。需要一种综合的方法来揭示膜融合的机制，从了解单个HA是如何干扰膜的水平到绘制整个病毒粒子-膜融合复合体的结构图。在这个计划中，ECT将被用来对整个病毒粒子和靶膜之间的预融合、半融合和融合孔的三维结构进行成像。将选择靶膜来测试脂质成分、小叶分布和几何形状在融合过程中的作用。我们还将使用ECT来分析可溶性HA胞外区和全长HA的膜弯曲活性。SAXS和HD-MS将结合起来确定融合基因、发夹前HA中间态的结构。该模型将通过化学交联和质谱分析进行验证。

DESCRIPTION (provided by applicant): Protein-mediated membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. Enveloped viruses use specialized protein machinery to fuse their membrane with the membrane of host cells and deliver their genetic material for replication. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike is responsible for host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the states that drive the fusion process have proven to be refractory to classical structure determination. In addition, the nature of membrane deformations during fusion has largely eluded characterization. In essence, we lack a mechanistic understanding of membrane fusion and hence viral entry. This proposal focuses on determining the structural and functional interplay between hemagglutinin and membranes during fusion. A combined approach including fluorescence spectroscopy, electron cryo-tomography (ECT), small-angle X-ray scattering (SAXS) with 3-D shape reconstruction, and Hydrogen-Deuterium-exchange with mass spectrometry analysis (HD-MS) will be employed. An integrative approach is needed to reveal the mechanics of membrane fusion from the level of understanding how individual HA spikes perturb membranes to mapping the architecture of whole virion-membrane fusion complexes. In this proposal, ECT will be used to image the 3-dimensional architecture of pre-fusion, hemi-fusion and fusion pores between whole virions and target membranes. Target membranes will be chosen to test the role of lipid composition, leaflet distribution, and geometry on the fusion process. We will also use ECT to dissect the membrane-bending activity of soluble HA ectodomain and full-length HA. SAXS and HD-MS will be combined to determine the structure of the fusogenic, prehairpin HA intermediate state. The model will be validated by chemical crosslinking with mass spectrometry analysis.