Dissecting Interactions between RNA and Coronavirus Nucleocapsid Proteins Using Native Mass Spectrometry

The nucleocapsid protein is the most abundant protein in the SARS-CoV virus and plays critical roles in RNA packaging and storage, yet the nature of the protein’s interactions with RNA remain elusive owing to the presence of multiple disordered regions of the protein. Here we evaluate the protein–RNA and protein–protein interactions of nucleocapsid proteins from the SARS-CoV-1 WT, SARS-CoV-2 WT, and SARS-CoV-1 omicron to better understand how the interactions are modulated by sequence variations. By using native mass spectrometry, we reveal that all three nucleocapsid proteins exist as both monomers and dimers that bind up to two RNA molecules per monomer, suggesting dual RNA binding sites. Ultraviolet photodissociation (UVPD) was used to localize the RNA binding sites found in the RNA binding domain and N-terminus of the nucleocapsid protein. The stabilities of the dimer complexes of the three nucleocapsid were monitored using energy-variable collision-induced dissociation, revealing that the dimer from SARS-CoV-1 is less stable than the dimers of the other two SARS-CoV variants in the gas phase. Furthermore, variable temperature electrospray ionization (ESI) experiments of the RNA-bound nucleocapsid proteins indicated that the protein–RNA interactions are stronger than the protein–protein interactions in solution as the dimeric protein–RNA complexes dissociate into monomeric protein–RNA complexes prior to the loss of RNA.