Interplay of protein and membrane intermediates during influenza virus fusion

描述（由申请人提供）：蛋白质介导的膜融合是一种基本的生物学过程，是包膜病毒感染、突触信号传导、胞内囊泡运输、配子受精和细胞-细胞融合的核心。尽管深入的研究，目前我们有一个有限的机制了解融合蛋白机械如何操纵脂膜，以诱导它们的融合。包膜病毒使用专门的蛋白质机制将其膜与宿主细胞膜融合，并传递其遗传物质进行复制。在流感病毒中，三聚体血凝素（HA）糖蛋白刺突负责宿主细胞附着和膜融合。虽然已经表征了融合机器的构象和部分的子集的结构，但是驱动融合过程的状态已经被证明是经典结构测定的难治性。此外，融合过程中膜变形的性质在很大程度上回避了表征。从本质上讲，我们缺乏对膜融合和病毒进入的机制的理解。该建议的重点是确定融合过程中血凝素和膜之间的结构和功能相互作用。将采用包括荧光光谱法、电子冷冻断层扫描（ECT）、小角X射线散射（SAXS）与三维形状重建以及氢氘交换与质谱分析（HD-MS）的组合方法。需要一种综合的方法来揭示膜融合的机制，从理解单个HA尖峰如何扰乱膜到绘制整个病毒体-膜融合复合物的结构的水平。在该提议中，ECT将用于对整个病毒体和靶膜之间的融合前、半融合和融合孔的三维结构进行成像。选择靶膜以检测脂质组成、瓣叶分布和几何形状对融合过程的作用。我们还将使用ECT来剖析可溶性HA胞外域和全长HA的膜弯曲活性。将结合SAXS和HD-MS来确定促融合的前发夹HA中间状态的结构。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.