Dual functions of the SARS-CoV-2 nucleocapsid domains mediate RNA binding and phase separation.

Abstract

The primary purpose of the nucleocapsid (N) protein of the Sars-CoV-2 coronavirus is to interact with viral RNA and drive the formation of the viral capsid, mediated by multivalent interaction of both the N- and C-terminal domains (NTD and CTD) of N with RNA. N additionally forms liquid-liquid phase separated droplets with RNA, both in vitro and in infected cells, in a process believed to be important to viral transcription and replication. The degree to which N phase separates with RNA is dependent on the specific RNA being tested, suggesting that the secondary structure of the RNA plays a role in the propensity for phase separation. The ∼32 kb CoV-2 genomic RNA contains a heterogeneous mix of paired and unpaired bases, and while studies suggest that paired RNA is the primary driver of phase separation, little is known at the structural level about how different RNA structures interact with the nucleocapsid. Here we present investigation of binding between N and short designer oligos built to be homogenously single-stranded or double-stranded (ssRNA or dsRNA). Interestingly, we find that our 14-mer dsRNA phase separates readily with the nucleocapsid, while the ssRNA does not, indicating underlying structural differences between the two interactions. Further, we demonstrate by nuclear magnetic resonance (NMR) spectroscopy, fluorescence anisotropy and gel shift assays, that the NTD binds preferentially to ssRNA, and the CTD to dsRNA. We show that the NTD interacts nonspecifically with dsRNA, and that mutations in the NTD that disrupt RNA binding promote this nonspecific interaction. Lastly, we demonstrate that the tendency of N domains to phase separate correlates with weak, nonspecific binding interactions with RNA, suggesting that this weak, nonspecific binding is the primary driver of phase separation.