The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveals a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.

This work was supported by the COVID research grant COV20/01265 awarded to M.V. from the Instituto de Salud Carlos III (ISCIII; Spain) and by the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). X-ray diffraction data collection was supported by the Spanish Synchrotron Radiation Facility ALBA through the COVID Proposal 2020074407 awarded to J.R.C.-T. and M.V.

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