BIOPHYSICS OF INFLUENZA HEMAGGLUTININ-MEDIATED FUSION

膜融合是一个普遍存在的细胞过程，但负责融合的蛋白质仅在包膜病毒的情况下被明确地确定。对于包膜病毒，细胞感染是由膜融合开始的。融合孔形成并扩大，病毒基因组通过孔进入细胞质。与其他融合蛋白相比，流感病毒血凝素（HA）融合的生物物理机制研究更为广泛。由于HA和包括HIV-1在内的许多其他病毒融合蛋白具有相同的核心结构，并且所有病毒融合蛋白都通过将融合肽插入膜中启动其作用，因此HA诱导融合的总体机制对于许多（如果不是全部）病毒融合蛋白来说可能是相似的。融合，包括孔隙行为，已经通过在细胞表面表达HA并将这些细胞融合到靶膜上进行了最广泛的研究。然而，细胞蛋白可以改变融合过程并影响毛孔。因此，单个流感病毒粒子将融合到不含蛋白质的磷脂双层膜上，导致融合孔形成的步骤、孔本身及其随后的扩大将通过电容测量来表征。通过确定荧光脂质染料能否通过最初形成的小融合孔，可以评估膜间是否在病毒融合后立即建立完全脂质连续性。通过蛋白水解去除病毒包膜中的透明质酸，可以系统地降低其密度，并测量其动力学，这样就可以估计出在孔形成过程中相关的透明质酸分子的数量。从结合状态到融合状态的蛋白质构象和脂质单层排列的中间状态已经被推断为表达HA的细胞系统。这些中间状态是否确实先于病毒系统的融合将被确定。将构建一个实验可测试的理论模型，将已知的HA在触发融合时所经历的结构变化与膜单分子层被认为进行的配置联系起来。给定的HA变化是否会导致相应的单层配置变化将通过显式计算确定。

Membrane fusion is a ubiquitous cellular process, but the proteins responsible for fusion have been unambiguously identified only in the case of enveloped virus. For enveloped virus, infection of cells is initiated by membrane fusion. A fusion pore forms and enlarges and the viral genome passes through the pore and into cytosol. The biophysical mechanism of fusion has been more extensively studied for hemagglutinin (HA) of influenza virus than for any other fusion protein. As HA and many other viral fusion proteins, including that of HIV-1, have the same core structure, and all viral fusion proteins initiate their action by insertion of fusion peptides into membranes, the overall mechanism by which HA induces fusion is probably similar for many, if not all, viral fusion proteins. Fusion, including pore behavior, has been most extensively studied by expressing HA on cell surfaces and fusing these cells to target membranes. However, cellular proteins could alter the fusion process and affect the pores. Individual influenza virions will therefore be fused to phospholipid bilayer membranes, which are free of protein, and the steps leading up to the formation of the fusion pore, the pore itself, and its subsequent enlargement will be characterized by electrical capacitance measurements. Whether full lipid continuity between membranes is established immediately upon fusion of a virus will be assessed by determining if fluorescent lipid dye can pass through the small fusion pore that initially forms. The density of HA in the viral envelope will be systematically reduced by proteolytically removing it and kinetics will be measured so that the number of HA molecules that associate in the creation of a pore can be estimated. Intermediate states of protein conformation and lipid monolayer arrangement from the bound state to fusion have been inferred for cellular systems that express HA. Whether these intermediate states do in fact precede fusion in the viral system will be established. An experimentally testable theoretical model will be constructed that relates the structural changes HA is known to undergo when fusion is triggered to the configurations through which membrane monolayers are thought to proceed. Whether a given change in HA can cause a corresponding change in monolayer configuration will be established by explicit calculation.