Structural and functional analysis of the coronavirus spike protein fusion peptide

冠状病毒刺突蛋白融合肽的结构和功能分析

• 批准号: 9761449
• 负责人: Susan Daniel
• 金额: $ 52.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-09 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761449
• 关键词: Animals; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell fusion; Cell membrane; Cell surface; Cells; Charge; Chimeric Proteins; Cleaved cell; Communicable Diseases; Complex; Coronavirus; Coronavirus Infections; Coronavirus spike protein; Cryoelectron Microscopy; Data; Disease Outbreaks; Ebola virus; Electron Spin Resonance Spectroscopy; Endosomes; Environment; Event; Exposure to; Family; Goals; HIV; Health; Human; Hydrophobicity; Infection; Influenza Hemagglutinin; Ions; Knowledge; Lead; Lipid Bilayers; Lipid Binding; Lipids; Mediating; Membrane; Membrane Fusion; Methods; Middle East Respiratory Syndrome Coronavirus; Modeling; Monitor; Mutagenesis; NMR Spectroscopy; Nature; Pathogenesis; Pathway interactions; Peptide Hydrolases; Peptides; Play; Population; Process; Property; Proteins; Reaction; Regulation; Research Personnel; Resources; Role; Route; Severe Acute Respiratory Syndrome; Site; Spectrum Analysis; Structure; Structure-Activity Relationship; Techniques; Testing; Tissues; Tropism; Viral; Viral Fusion Proteins; Viral Pathogenesis; Virus; Virus Receptors; Zoonoses; base; biophysical techniques; cell type; experimental study; flexibility; fluorescence microscope; in vivo; innovation; medical countermeasure; mutant; novel; novel virus; particle; pathogen; peptide I; peptide structure; protein function; receptor; receptor binding; structural biology; transmission process; virus envelope

Project Summary / Abstract Enveloped viruses access their host cells by binding to receptors on the plasma membrane and then undergoing fusion with the host membrane. Both binding and fusion are mediated by a specific viral “spike” protein that is typically primed for fusion activation by proteolytic cleavage to expose the fusion peptide. Coronavirus fusion spike protein (CoV S) is a complex biomolecular machine that has a novel fusion peptide with has a great deal of inherent flexibility in its fusion reaction. This is exploited by these viruses in their diverse entry pathways and is a primary determinant of viral tropism. We have pioneered the concept that that the proteolytic cleavage events in S that lead to membrane fusion occur both at the interface of the receptor binding (S1) and fusion (S2) domains (called S1/S2), as well as adjacent to a structurally and functionally novel fusion peptide within S2 (called S2’). Thus, there are notable differences between CoV S and most other class I fusion proteins including: 1) that the proteolytic events liberating the fusion peptide are diverse, and 2) that the fusion peptide itself is atypical in sequence compared to other fusion peptides, containing a mixture of important hydrophobic and negatively- charged residues, and may represent a larger than normal fusion “platform” instead of a defined “peptide”. Thus fusion peptide activity is likely controlled by reorganization of the fusion platform, based on both hydrophobic (i.e. lipid-binding) and ionic (i.e. Ca2+) interactions. Despite the recent availability of S structures in their pre- fusion state, there remains a very limited mechanistic understanding of membrane fusion for the CoV family, or any structural information to correlate structural biology aspects of S to its function in membrane fusion. This information is critical to understanding viral pathogenesis and CoV emergence into the human population. We propose an integrated biophysical, biochemical, and in vivo approach to study the unique cleavage-activated regulation of CoV S protein, using Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) as primary models. We will use state-of-the-art spectroscopy and an innovative single particle tracking technique to study S protein fusion peptide function, and combine these with in vivo infectivity studies, including at BSL3, will allow a complete picture of CoV fusion activation. These approaches will reveal how structure and function vary depending on the key activators of S; i.e. receptor binding, protease availability and the local ionic environment. These studies will allow us to determine common principals that can be applied to all CoVs, moving the field forward with these innovative studies will provide critical knowledge about CoV entry and tropism needed to safeguard human health from an emerging pathogen likely to cause severe outbreaks, and for which few or no medical countermeasures exist.

项目摘要 /摘要 包裹的病毒通过与质膜上的受体结合，然后经历。 与宿主膜融合。结合和融合都由特定的病毒“尖峰”蛋白介导 通常通过蛋白水解裂解以暴露融合肽的融合激活。冠状病毒融合 尖峰蛋白（COV S）是一款复杂的生物分子机器，具有新型的融合肽的含量很大 其融合反应中固有的灵活性。这些病毒在其潜水者的进入途径中探讨了这一点， 是病毒性偏向主义的主要决定者。我们开创了蛋白水解裂解事件的概念 在导致膜融合的S中，同时出现在接收器结合（S1）和融合（S2）域的界面上 （称为S1/S2），以及与S2内的结构和功能新颖的融合肽相邻（称为S2'）。 那就是COVS和大多数其他I类融合蛋白之间存在显着差异，包括：1） 释放融合肽的蛋白水解事件是潜水员，2）融合肽本身在 与其他融合宠物相比，序列含有重要的疏水性和负面的混合物 带电的残留物，可能代表比正常的融合“平台”，而不是定义的“肽”。那 融合肽活性可能通过融合平台的重组来控制，这是基于两个疏水性的 （即脂质结合）和离子（即Ca2+）相互作用。尽管最近在其前的s结构可用 融合状态，对COV家族的膜融合的机械理解仍然非常有限，或 任何结构信息将S的结构生物学方面与其在膜融合中的功能相关联。这 信息对于理解病毒发病机理和COV出现至关重要。我们 提案一种综合生物物理，生化和体内方法，用于研究独特的切割激活 使用中东呼吸综合征冠状病毒（MERS-COV）和严重的急性调节COV的蛋白质 呼吸综合征冠状病毒（SARS-COV）作为主要模型。我们将使用最先进的光谱 以及一种创新的单粒子跟踪技术，用于研究S蛋白融合肽功能，并结合这些 随着体内感染研究（包括在BSL3），将允许完整的COV融合激活图。这些 方法将揭示结构和功能如何根据S的关键激活因素而变化；即受体结合， 蛋白酶的可用性和局部离子环境。这些研究将使我们能够确定共同的校长 可以应用于所有COV，通过这些创新研究将领域向前发展，将提供关键 关于COV进入和潮流的了解，以保护人类健康免受新兴病原体的影响 引起严重的爆发，并且很少有医疗对策。