Structure, dynamics and variation in influenza hemagglutinin-driven fusion

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。长期以来，导致融合的融合蛋白和膜的事件顺序和机制相互作用一直抵制结构表征，但通过使用冷冻电子断层扫描(Cryo-ET)和结构质谱学等技术，我们可以在接近生理条件下研究天然HA和完整病毒，最近我们在连接结构、生物物理机制和生物功能方面取得了重大进展。我们最初的研究集中在特征良好的X31 H3N2重排病毒株上。这项工作揭示了HA融合蛋白上激活热点的性质，并阐明了导致高效融合的HA驱动的膜重塑的结构和序列。目前提出的项目的目标是通过比较不同亚型的HA和携带促进流感病毒传播到新宿主的突变的HA，应用这些强大的方法来表征HA介导膜融合的功能变异。我们还将研究颗粒形态变化对融合过程的影响，因为M1基质蛋白与HA一起介导融合，它也是病毒粒子形态的主要决定因素。从机制上理解HA/M1介导的融合中的变异，并与结构框架相联系，将为理解流感病毒的致病性、传播和进化提供有价值的基础。反过来，这些信息可以提供一个独特的视角来帮助指导流感疫苗结构的合理选择、免疫原的开发和HA靶向抑制剂的设计。

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. The sequence of events and mechanistic interplay of fusion protein and membranes that lead to fusion have long resisted structural characterization, but through the use of techniques such as cryo-electron tomography (cryo-ET) and structural mass spectrometry that allow native HA and intact virus to be studied under near physiological conditions, we have recently made significant progress in connecting structure, biophysical mechanisms and biological function. Our initial studies focused on the well-characterized X31 H3N2 reassortant virus strain. This work revealed the nature of activation hotspots on the HA fusion protein and elucidated the architecture and sequence of HA-driven membrane remodeling that leads to efficient fusion. The goal of the current proposed project is to apply these powerful approaches to characterize functional variation in HA-mediated membrane fusion by comparing HA from different subtypes and bearing mutations that promote transmission of influenza virus to new hosts. We also will examine the effect of variation in particle morphology on the fusion process, since the M1 matrix protein works together with HA to mediate fusion and it is also the primary determinant of virion morphology. A mechanistic understanding of variations in HA/M1-mediated fusion, linked to a structural framework, will provide a valuable basis for understanding influenza virus pathogenicity, transmission, and evolution. In turn, this information can offer a unique perspective to help guide rational selection of influenza vaccine constructs, immunogen development, and design of HA-targeting inhibitors.