Thursday, November 21, 2024
11/21/2024
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.
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