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

SARS-CoV-2是一场前所未有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多的蛋白质，在基因组衣壳化和病毒组装中起着至关重要的作用。然而，到目前为止，N还没有对其全长分子进行结构测定。事实上，目前还没有任何冠状病毒的全长N的高分辨率结构，尽管N内的单个结构域存在多个结构。缺乏关于N的结构信息，其组装和相互作用以及基因组的封装源于三个内在无序区域所带来的固有灵活性。在之前的工作中，在没有RNA或存在来自表达系统的随机细菌RNA的情况下，N过于灵活，无法确定高分辨率的结构。病毒粒子中组装的衣壳在灵活性、位置和构象上也太不均匀，无法提供高分辨率的信息。通过使用电迁移位移试验、尺寸排除和电子显微镜筛选进行仔细分析，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，可以进行高分辨率结构分析，并且代表了基本构建块和可能的组装中间体完整的衣壳。我们进一步在体外制备了一种聚合全长衣壳，也可用于结构研究。在这里，我们提出了二聚体、组装中间体和全长体外衣壳的低温电子显微镜，辅以创新的天然质谱法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）发生在组装、蛋白质-蛋白质和蛋白质- rna相互作用位点的构象调整；（iv）可能是抗病毒发展的目标位点。

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.