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: 10222497
• 负责人: Susan Daniel
• 金额: $ 43.97万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222497
• 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; 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; SARS coronavirus; 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; emerging pathogen; experimental study; flexibility; fluorescence microscope; in vivo; innovation; medical countermeasure; mutant; novel; novel coronavirus; novel virus; particle; 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.

项目概要/摘要 有包膜病毒通过与质膜上的受体结合来进入宿主细胞，然后经历 与宿主膜融合。结合和融合均由特定的病毒“刺突”蛋白介导，该蛋白是 通常通过蛋白水解裂解暴露融合肽来进行融合激活。冠状病毒融合 刺突蛋白（CoV S）是一种复杂的生物分子机器，具有一种新型融合肽，具有多种功能。 其聚变反应固有的灵活性。这些病毒通过其不同的进入途径利用了这一点 是病毒趋向性的主要决定因素。我们率先提出了这样的概念：蛋白水解裂解事件 导致膜融合的 S 发生在受体结合 (S1) 和融合 (S2) 结构域的界面 （称为 S1/S2），以及邻近 S2 内结构和功能新颖的融合肽（称为 S2'）。 因此，冠状病毒 S 与大多数其他 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多种多样的，并且 2) 融合肽本身是非典型的 与其他融合肽相比，该序列包含重要的疏水性和负性的混合物 带电残基，并且可能代表比正常融合“平台”更大的融合“平台”而不是定义的“肽”。因此 融合肽活性可能是通过基于疏水性的融合平台的重组来控制的 （即脂质结合）和离子（即 Ca2+）相互作用。尽管最近S结构在其预制中可用 融合状态，对 CoV 家族膜融合的机制了解仍然非常有限，或者 将 S 的结构生物学方面与其在膜融合中的功能相关联的任何结构信息。这 信息对于了解病毒发病机制和冠状病毒在人群中的出现至关重要。我们 提出了一种综合的生物物理、生物化学和体内方法来研究独特的裂解激活 利用中东呼吸综合征冠状病毒 (MERS-CoV) 和严重急性呼吸综合征冠状病毒 S 蛋白的调节 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学 以及创新的单粒子追踪技术来研究S蛋白融合肽的功能，并将这些结合起来 通过体内感染性研究（包括 BSL3），将能够全面了解 CoV 融合激活情况。这些 方法将揭示结构和功能如何根据 S 的关键激活剂而变化；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原则 可以应用于所有冠状病毒，通过这些创新研究推动该领域的发展将提供关键的 保护人类健康免受新出现的病原体侵害所需的关于冠状病毒进入和趋向性的知识 导致严重的疫情暴发，而对此几乎没有或根本没有医疗对策。