Structure of the SARS-CoV-2 Nucleocapsid: building block to viral capsid

SARS-CoV-2 核衣壳的结构：病毒衣壳的构建模块

• 批准号: 10728253
• 负责人: Erica Ollmann Saphire
• 金额: $ 28.59万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-05 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10728253
• 关键词: 2019-nCoV; Address; Affinity; Antibodies; Bacterial RNA; Biochemical; Biological Assay; Capsid; Cells; Complement; Complex; Coronavirus; Cryoelectron Microscopy; Data; Diameter; Dimerization; Disease; Electron Microscopy; Electrophoretic Mobility Shift Assay; Exclusion; Family; Genetic Transcription; Genome; Goals; Hand; Histones; In Vitro; Individual; Label; Length; Maps; Mass Spectrum Analysis; Mediating; Medicine; Modeling; Molecular; Molecular Conformation; Molecular Machines; Nucleocapsid; Nucleocapsid Proteins; Play; Polymers; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Viruses; RNA chemical synthesis; RNA-Protein Interaction; Resolution; Role; SARS-CoV-2 genome; SARS-CoV-2 inhibitor; SARS-CoV-2 variant; Site; Structural Protein; Structure; System; Viral; Viral Structural Proteins; Virion; Virus; Virus Assembly; Visualization; Work; antiviral drug development; coronavirus antiviral; crosslink; density; dimer; flexibility; innovation; pandemic disease; particle; polymerization; rational design; reconstitution; screening; self organization; stem; stoichiometry; structural biology; variants of concern; viral RNA

SARS-CoV-2, the causative agent of an unprecedented global pandemic, has just four structural proteins. Of these, the nucleocapsid N is the most abundant protein in the virion, and plays essential roles in genome encapsidation and viral assembly. However, N has thus far defied structure determination of its full-length molecule. Indeed, there are currently no high-resolution structures of full-length N for any coronavirus, although multiple structures exist for individual domains within N. The lack of structural information on N, its assembly and its interactions and encapsidation of the genome stem from the inherent flexibility contributed by three intrinsically disordered regions. In previous work, N, in the absence of RNA or in the presence of random bacterial RNA derived from the expression system, was too flexible to allow determination of a high-resolution structure. The assembled capsid in the virion is also too heterogeneous in its flexibility, positions and conformations to afford high-resolution information. Through careful analysis using electromobility shift assays, size-exclusion and screening by electron microscopy, we have now identified portions of the SARS-CoV-2 genome that yield structurally homogeneous, purified N dimers, octamers, and 16-mers that are amenable to high-resolution structural analysis, and which represent the basic building block and likely assembly intermediates of the full capsid. We have further produced a polymerized full-length capsid in vitro that is also amenable to structural study. Here we propose cryoEM of the dimer, assembly intermediates and full length in vitro capsid, complemented by innovative native mass spectrometry and straightforward specific antibody-mediated domain identification. This work will illuminate (i) the structure and assembly of the coronavirus capsid; (ii) how the RNA genome interacts with multiple domains of the full- length N and connects along polymerized copies of N; (iii) conformational adjustments that occur in assembly, protein-protein and protein-RNA interaction sites; and (iv) sites that may be amenable targets for antiviral development.

SARS-CoV-2是一种前所未有的全球大流行病的病原体，它只有四种结构蛋白。其中，核衣壳N是病毒粒子中最丰富的蛋白质，在基因组壳体化和病毒组装中发挥着重要作用。然而，迄今为止，N一直无法确定其全长分子的结构。事实上，目前还没有任何冠状病毒全长N的高分辨率结构，尽管N内的单个结构域存在多种结构。缺乏N的结构信息，它的组装和它的相互作用和基因组的重叠源于三个固有的无序区域所贡献的固有的灵活性。在以前的工作中，N，在RNA的情况下，或在存在的随机细菌RNA来自表达系统，是太灵活，允许确定一个高分辨率的结构。病毒体中组装的衣壳在其灵活性、位置和构象方面也过于异质，无法提供高分辨率信息。通过使用电迁移率变化分析，尺寸排阻和电子显微镜筛选的仔细分析，我们现在已经确定了SARS-CoV-2基因组的部分，这些部分产生结构均匀，纯化的N二聚体，八聚体和16聚体，这些都适合高分辨率结构分析，并且代表了完整衣壳的基本构建模块和可能的组装中间体。我们已经进一步在体外产生了聚合的全长衣壳，其也适合于结构研究。在这里，我们提出了二聚体，组装中间体和全长体外衣壳的cryoEM，辅以创新的本地质谱和简单的特异性抗体介导的结构域鉴定。这项工作将阐明（i）冠状病毒衣壳的结构和组装;（ii）RNA基因组如何与全长N的多个结构域相互作用并沿N的沿着聚合拷贝连接;（iii）在组装、蛋白质-蛋白质和蛋白质-RNA相互作用位点中发生的构象调整;以及（iv）可能成为抗病毒开发的顺从靶点的位点。