Influenza Hemagglutinin: Structure, Dynamics, and Cooperativity During Fusion

描述（由申请人提供）项目概述：甲型流感病毒是理解病毒通过受体介导的内吞作用进入宿主细胞的范例。我们试图加深我们对病毒膜融合的理解，以确定可能的策略来干扰这一过程并阻止感染周期。血凝素（HA）蛋白介导病毒和宿主细胞膜的融合。HA与其他1型融合蛋白（如在埃博拉病毒和HIV中发现的那些）共享核心元件。虽然HA介导的融合的某些方面已经被表征，但在实际驱动膜融合的分子构象的表征中，在我们对HA如何改变构象的理解中，以及在我们对融合蛋白和膜变形之间的相互作用的理解中，仍然存在显著的差距。我们建议使用溶液X射线和中子散射从头形状重建，以确定分离的HA和HA与脂质双层相互作用的血凝素组装的结构和动力学。通过对接已知的高分辨率结构的组件到解决方案散射衍生的形状信封，赝原子模型将组成。这种类型的综合方法是必要的表征复杂的大分子机器。实验将提供独特的洞察力的构象轨迹穿越HA，因为它从亚稳态到融合活性形式转换，确定是否膜结合HA表现出组装成孔复合物的倾向，并阐明膜结构的变化。此外，单颗粒荧光显微镜将用于揭示HA在病毒膜上排列时的功能背景。这种方法将确定病毒表面上的数百个HA刺突是否以合作的方式变得具有融合活性。 相关性：流感病毒是一种主要的人类病原体，在典型的一年中感染5-20%的美国人口，并导致超过200，000人住院和约36，000人死亡。这项拟议中的研究旨在详细了解流感用来入侵新宿主细胞的血凝素机制的功能。这样的理解是必要的，以确定如何最好地干扰机制，并阻止病毒感染的周期。

DESCRIPTION (provided by applicant) Project Summary: Influenza A virus is a paradigm for understanding viral entry of host cells via receptor-mediated endocytosis. We seek to deepen our understanding of viral membrane fusion in order to identify possible strategies for interfering with the process and arresting the infection cycle. The hemagglutinin (HA) protein mediates fusion of the virus and host cell membranes. HA shares core elements with other type-1 fusion proteins such as those found in Ebola virus and HIV. While certain facets of HA-mediated fusion have been characterized, significant gaps remain in the characterization of the molecular conformations that actually drive membrane fusion, in our understanding of how HA changes conformation, and in our understanding of the interplay between fusion protein and membrane deformation. We propose to use solution X-ray and neutron scattering with ab initio shape reconstruction to determine the structure and dynamics of the hemagglutinin assembly both for isolated HA and HA that is interacting with lipid bilayers. By docking known high resolution structures of components into the solution scattering-derived shape envelopes, pseudoatomic models will be composed. This type of integrative approach is necessary for characterization of complex macromolecular machines. The experiments will provide unique insight into the conformational trajectory traversed by HA as it converts from metastable to fusion-active form, determine whether membrane-bound HA exhibits a propensity to assemble into pore complexes, and elucidate resultant changes in membrane structure. In addition, single-particle fluorescence microscopy will be used to reveal the functional context of HA when it is arrayed on the viral membrane. This approach will identify whether the hundreds of HA spikes on the virus surface become fusion-active in a cooperative fashion. Relevance: Influenza virus is a major human pathogen that in a typical year infects 5-20% of the population of the United States and results in more than 200,000 hospitalizations and approximately 36,000 deaths. The proposed research seeks to gain a detailed understanding of the function of the hemagglutinin machinery that influenza uses to invade new host cells. Such an understanding is necessary in order to identify how best to interfere with the mechanism and to arrest the cycle of viral infection.