SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity. SYNOPSIS image Viruses can assemble nucleocapsid protein (N) and genomic RNA in dynamic compartments containing host ribonucleoproteins. We show SARS-CoV-2 N undergoes liquid-liquid phase separation (LLPS) with RNA and enters droplets formed by human hnRNPs.N phase separation in vitro in physiological buffer conditions is enhanced by RNA. N-terminal and linker disordered domains and C-terminal folded dimerization domain are essential for robust LLPS. RNA sequence specificity is not necessary for enhancing N LLPS. N partitions into phase separated forms of hnRNPA2, TDP-43, and FUS.