Influenza Hemagglutinin: Structure, Dynamics, and Cooperativity During Fusion

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，解决方案X射线散射从头形状重建，以确定的结构和动力学的血凝素融合蛋白。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。此外，冷冻电子断层扫描将用于阐明HA介导的融合孔的细胞和蛋白质结构，并确定排列在膜上的HA的高级组织。最后，单颗粒荧光显微镜将用于揭示HA在病毒膜上排列时的动态融合-激活。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。

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 ceil 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 scattering with ab initio shape reconstruction to determine the structure and dynamics of the hemagglutinin fusion protein. 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. In addition, cryo-eiectron tomography will be used to elucidate the lipidic and proteinaceous architecture of the HAmediated fusion pore and to determine the higher-order organization of HA arrayed on membranes. Lastly, single-particle fluorescence microscopy will be used to reveal the dynamic fusion-activation 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.