RNA-Binding Specificity of the SARS-CoV-2 Nucleocapsid Protein is Determined by Binding Kinetics of the N-Terminal Domain to ssRNA

SummaryThe SARS-CoV-2 nucleocapsid phosphoprotein (CoV-N) contains two structured RNA binding domains, the NTD and the CTD, but the functional implications of two structurally independent binding domains remains unclear. The NTD/RNA interaction is well described but information about CTD/RNA binding is more limited. With a 1000 nucleotide fragment (g1-1000) of the SARS-CoV-2 genomic RNA, we map the interactions of both domains using NMR and show that CTD is sufficient for droplet formation. Further, we investigate CoV-N interactions with a primary RNA-binding site, g74-301, and a shuffled version with fewer paired nucleotides and higher entropy, sh-g74-301. Probed by microscopy and SEC-MALS, CoV-N forms droplets and large complexes faster with sh-g74-301. NMR titrations and mutagenesis studies confirm that NTD is responsible for the difference in binding patterns. These results suggest that CoV-N has preference for ssRNA, and that binding specificity is determined by NTD binding kinetics, while CTD is necessary for droplet formation.Funding: This work is funded by an NSF EAGER MCB 2034446. We also acknowledge the support of the Oregon State University NMR Facility funded by the National Institutes of Health, HEI Grant 1S10OD018518, and the M. J. Murdock Charitable Trust grant #2014162.Declaration of Interests: None to declare.