Biophysical studies of viral membrane fusion proteins

病毒膜融合蛋白的生物物理学研究

• 批准号: 10642837
• 负责人: James B Munro
• 金额: $ 50.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642837
• 关键词: 2019-nCoV; Address; Adopted; Adoption; Apical; Avian Influenza; Avian Influenza A Virus; Binding; Binding Sites; Biological Assay; Biological Models; Biophysics; Birds; Cell surface; Cells; Cellular Membrane; Chimeric Proteins; China; Cryo-electron tomography; Data; Development; Dissociation; Distal; Drug Targeting; Endocytosis; Ensure; Environment; Equilibrium; Evolution; Fluorescence Resonance Energy Transfer; Foundations; Glycoproteins; Goals; Hemagglutinin; Human; Image; In Vitro; Influenza; Influenza A virus; Influenza Hemagglutinin; Kinetics; Life Cycle Stages; Link; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Modeling; Molecular; Molecular Conformation; Morphology; Mutation; Pathway interactions; Peptides; Phenotype; Population; Public Health; Publishing; Research; Role; SARS-CoV-2 spike protein; Sialic Acids; Specificity; Structure; Surface; Testing; Therapeutic; Thermodynamics; Viral; Viral Fusion Proteins; Virion; Virus; Visualization; Zoonoses; biophysical analysis; biophysical properties; cold temperature; combat; data exchange; density; emerging pathogen; endosome membrane; env Gene Products; experimental study; influenza infection; influenza virus strain; inhibitor; late endosome; molecular dynamics; next generation; pandemic disease; pandemic potential; premature; prevent; receptor binding; sialic acid receptor; single molecule; tissue tropism; trafficking; transmission process; viral entry inhibitor

SUMMARY Influenza A virus (IAV) hemagglutinin (HA) is the canonical example of a class-I viral fusion protein, and thus provides an ideal model system for understanding the fusion mechanisms of many different viruses. Numerous other viral envelope glycoproteins, including the SARS-CoV-2 spike, are believed to mediate fusion by a comparable mechanism. Our long-term goal is to establish a complete mechanistic framework of class-I viral fusion. We further aim to identify conserved and divergent features of the fusion mechanisms of distinct viruses, generating specific models that fit within the general framework. HA resides on the surface of the IAV virion and facilitates attachment to the target cell surface through the receptor-binding domain (HA1) engaging SA moieties. Following endocytosis and trafficking to the late endosome, HA promotes fusion of the viral and endosomal membranes. The model of HA-mediated membrane fusion describes a “spring-loaded” metastable prefusion conformation at neutral pH. Dissociation of HA1 from the fusion domain (HA2) allows HA2 to undergo a cascade of conformational changes that drive membrane fusion. While extensive structural data exist for HA pre- and postfusion, and alternative conformations have been visualized and inferred, the conformational trajectory that leads to membrane fusion, including the adoption of anticipated intermediates, has never been explicitly validated. Nor has the order and timing of conformational changes and membrane fusion been determined. Here, we will utilize a multifaceted approach involving single-molecule Förster resonance energy transfer imaging, single-virion fusion, cryoelectron tomography, and molecular dynamic simulation to directly visualize the conformational trajectory undergone by HA during membrane fusion. We will explore the roles of virion morphology, HA density and cooperativity, and target membrane lipid content in mediating HA conformational changes and the mechanism of fusion. We will describe the allosteric connection between distal regions of HA that regulate the timing of fusion, drawing comparison to SARS-CoV-2 S. Finally, we will biophysically characterize the phenotypic differences between human and avian IAV strains to determine what prevents avian IAV strains from entering the human population.

摘要 甲型流感病毒(IAV)血凝素(HA)是I类病毒融合蛋白的典型例子，因此 为了解多种不同病毒的融合机制提供了理想的模型系统。数不胜数 其他病毒包膜糖蛋白，包括SARS-CoV-2尖峰，被认为通过一种 类似的机制。我们的长期目标是建立一个完整的I类病毒的机制框架 核聚变。我们的进一步目标是确定不同病毒融合机制的保守和分歧特征， 生成符合一般框架的特定模型。HA存在于IAV病毒粒子的表面， 通过与SA部分结合的受体结合域(HA1)促进与靶细胞表面的附着。 在内吞和运输到晚期内体之后，HA促进病毒和内体的融合 膜。透明质酸介导膜融合模型描述了一种“弹簧加载”的亚稳态预融合。 中性pH条件下的构象。HA1与融合结构域(HA2)的解离允许HA2经历级联 驱动膜融合的构象变化。虽然目前已有大量的房委会前期和 融合后，并已可视化和推断出替代构象，构象轨迹 导致膜融合，包括采用预期的中间体，从未被明确 已验证。构象变化和膜融合的顺序和时间也没有确定。这里, 我们将利用一种多方面的方法，涉及单分子Förster共振能量转移成像， 单病毒粒子融合、低温电子断层扫描和分子动力学模拟，以直接可视化 HA在膜融合过程中的构象轨迹。我们将探索病毒粒子的作用 HA形态、密度和协作性以及靶膜脂含量对HA构象的影响 变化和融合的机制。我们将描述HA远端区域之间的变构连接 调节融合的时机，将其与SARS-CoV-2S进行比较。最后，我们将从生物物理学上 鉴定人和禽类IAV毒株之间的表型差异，以确定是什么阻止了禽类 IAV毒株进入人类人口。