BIOPHYSICS OF INFLUENZA HEMAGGLUTININ-MEDIATED FUSION

流感血凝素介导的融合的生物物理学

• 批准号: 2908298
• 负责人: FREDRIC S COHEN
• 金额: $ 3.96万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2908298
• 关键词: Orthomyxoviridae; artificial membranes; biophysics; chemical kinetics; conformation; electrophysiology; fluorescence resonance energy transfer; fluorescent dye /probe; lipid bilayer membrane; liposomes; membrane fusion; membrane structure; microorganism hemagglutinin; physical model; virus envelope; virus infection mechanism; virus protein; voltage /patch clamp

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.

膜融合是一种无处不在的细胞过程，但是仅在包膜病毒的情况下才明确鉴定了负责融合的蛋白质。 对于包膜病毒，细胞的感染是由膜融合引发的。 融合孔形成和扩大，病毒基因组通过孔进入细胞质。 与任何其他融合蛋白相比，对融合的生物物理机制已更广泛地研究了流感病毒的血凝素（HA）。 由于HA和许多其他病毒融合蛋白（包括HIV-1）具有相同的核心结构，并且所有病毒融合蛋白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可能与许多人可能相似，如果不是全部，则病毒融合蛋白。 通过在细胞表面上表达HA并将这些细胞靶向靶膜的融合，包括孔隙行为，最广泛地研究了融合。但是，细胞蛋白可以改变融合过程并影响孔。 因此，单个流感病毒体将融合到没有蛋白质的磷脂双层膜中，并且导致融合孔形成的步骤，孔本身，其后续膨胀将以电气测量为特征。 通过确定荧光脂质染料是否可以通过最初形成的小融合孔，可以评估膜之间的全脂质连续性是否立即建立。 通过将其去除蛋白水解，将系统地降低病毒包膜中HA的密度，并测量动力学，以便可以估计与创建孔相关的HA分子数量。 对于表达HA的细胞系统，已经推断出蛋白质构象和脂质单层排列的中间状态。 这些中间状态实际上是否会在病毒系统中建立融合之前。 将构建一个可以实验测试的理论模型，该模型将融合触发到膜单层可以进行的结构时，已知HA会发生HA。 HA中给定的更改是否会导致单层配置的相应变化，将通过显式计算建立。