Influenza Hemagglutinin: Structure, Dynamics, and Cooperativity During Fusion

甲型流感病毒是一种通过受体介导的内吞作用了解病毒进入宿主细胞的范例。 我们试图加深对病毒膜融合的理解，以确定干扰这一过程并阻止感染循环的可能策略。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(如埃博拉病毒和艾滋病毒中发现的融合蛋白)具有共同的核心成分。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、我们对HA如何改变构象的理解以及对融合蛋白和膜变形之间的相互作用的理解方面仍然存在着显著的差距。我们建议使用溶液X射线散射和从头算形状重建来确定血凝素融合蛋白的结构和动力学。通过将已知的高分辨率组分结构对接到溶液散射导出的形状包络中，将组成伪原子模型。这种综合方法对于表征复杂的大分子机器是必要的。这些实验将提供独特的洞察，当HA从亚稳态转变为融合活性形式时，它所经过的构象轨迹。此外，冷冻电子断层扫描将被用来阐明HA介导的融合孔的脂类和蛋白质结构，并确定排列在膜上的HA的高阶组织。最后，利用单粒子荧光显微镜观察了HA在病毒膜上的动态融合激活过程。这种方法将确定病毒表面的数百个HA尖峰是否以合作的方式变得融合活性。

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.