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.

总结 甲型流感病毒（IAV）血凝素（HA）是I类病毒融合蛋白的典型实例，因此 为理解多种不同病毒的融合机制提供了一个理想的模型系统。许多 其他病毒包膜糖蛋白，包括SARS-CoV-2刺突蛋白，被认为是通过一种 类似的机制。我们的长期目标是建立一个完整的机制框架， 核聚变我们进一步的目标是确定不同病毒融合机制的保守和不同特征， 生成适合一般框架的特定模型。HA存在于IAV病毒体的表面， 促进通过受体结合结构域（HA 1）接合SA部分附着到靶细胞表面。 在胞吞作用和运输到晚期内体之后，HA促进病毒和内体的融合。 膜。HA介导的膜融合模型描述了一个“弹簧加载”的亚稳态预融合 HA 1与融合结构域（HA 2）的解离允许HA 2经历级联反应， 导致膜融合的构象变化虽然存在大量的结构数据， 融合后，和替代构象已经可视化和推断，构象轨迹， 导致膜融合，包括采用预期的中间体，从未明确过 验证.也没有确定构象变化和膜融合的顺序和时间。在这里， 我们将利用涉及单分子Förster共振能量转移成像的多方面方法， 单病毒体融合，冷冻电子断层扫描和分子动力学模拟，以直接可视化 HA在膜融合过程中经历的构象轨迹。我们将探讨病毒粒子的作用 形态、HA密度和协同性以及介导HA构象形成的靶膜脂质含量。 变化和融合机制。我们将描述HA远端区域之间的变构连接 与SARS-CoV-2S进行比较，最后，我们将从生物制药学的角度 描述人类和禽类IAV毒株之间的表型差异，以确定预防禽流感的因素。 传染给人类。