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 是史无前例的全球大流行病的病原体，只有四种结构蛋白。其中，核衣壳 N 是病毒体中最丰富的蛋白质，在基因组衣壳化和病毒组装中发挥重要作用。然而，N 迄今为止仍无法确定其全长分子的结构。事实上，目前任何冠状病毒都没有全长 N 的高分辨率结构，尽管 N 内的各个结构域存在多种结构。N、其组装、相互作用以及基因组衣壳化的结构信息的缺乏源于由三个本质上无序的区域贡献的固有灵活性。在之前的工作中，在不存在 RNA 或存在来自表达系统的随机细菌 RNA 的情况下，N 过于灵活，无法确定高分辨率结构。病毒粒子中组装的衣壳在灵活性、位置和构象方面也过于异质，无法提供高分辨率信息。通过使用电动迁移率测定、尺寸排除和电子显微镜筛选进行仔细分析，我们现在已经鉴定出 SARS-CoV-2 基因组的部分，这些部分产生结构均质的、纯化的 N 二聚体、八聚体和 16 聚体，这些聚体适合高-分辨率结构分析，代表完整衣壳的基本构建块和可能的组装中间体。我们进一步在体外制备了聚合的全长衣壳，该衣壳也适合结构研究。在这里，我们建议对二聚体、组装中间体和全长体外衣壳进行冷冻电镜分析，并辅以创新的天然质谱分析和直接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着N的聚合拷贝连接； (iii) 组装、蛋白质-蛋白质和蛋白质-RNA 相互作用位点发生的构象调整； (iv) 可能成为抗病毒开发目标的位点。