Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation

Abstract

The nucleocapsid (N) protein of SARS-CoV-2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35-40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine (SR) region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.