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

SARS-CoV-2是一场史无前例的全球大流行的病原体，它只有四种结构蛋白。其中，核衣壳N是病毒粒子中含量最丰富的蛋白质，在基因组封装和病毒组装中起着至关重要的作用。然而，到目前为止，N一直无视其全长分子的结构确定。事实上，目前还没有针对任何冠状病毒的全长N的高分辨率结构，尽管N中的各个结构域存在多种结构。缺乏关于N及其组装、相互作用和基因组封装的结构信息源于三个内在无序区域所带来的固有灵活性。在以前的工作中，在没有RNA或存在来自表达系统的随机细菌RNA的情况下，N太灵活，无法确定高分辨率结构。病毒粒子中组装的衣壳在灵活性、位置和构象上也过于不同，无法提供高分辨率的信息。通过使用电子迁移率位移分析、大小排除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基因组的一部分，它们产生结构均一的、纯化的N二聚体、八聚体和16-MERS，这些都是高分辨率结构分析的结果，它们代表了整个衣壳的基本构件和可能的组装中间体。我们还在体外生产了一种聚合的全长衣壳，这也是结构研究的结果。在这里，我们提出了二聚体、组装中间体和体外全长衣壳的冷冻EM，并辅之以创新的自然质谱学和直接的特异性抗体介导域识别。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的聚合副本连接；(Iii)在组装、蛋白质-蛋白质和蛋白质-RNA相互作用部位发生的构象调整；以及(Iv)可能成为抗病毒开发的靶点。

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.