Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the N(IDR), the serine/arginine rich motif (SR(IDR)), and the C(IDR). Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid–liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (N(FL)) assembles into ring-like architecture whereas the truncated SR(IDR)-CTD-C(IDR) (N(182-419)) promotes filamentous assembly. Moreover, LLPS droplets of N(FL) and N(182-419) are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N(182-419) droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s43556-023-00129-z.