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

The nucleocapsid (N) protein of coronaviruses is responsible for compaction of the ~30-kb RNA genome in the ~100-nm virion. Cryo-electron tomography suggests 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 with viral RNA fragments in vitro, forms cylindrical 15-nm particles similar to the vRNP structures observed within coronavirus virions. These vRNPs form in the presence of stem-loop-containing RNA and 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 and disrupts vRNP assembly. We propose that unmodified N binds stem-loop-rich regions in genomic RNA to form compact vRNP complexes within the nucleocapsid, while phosphorylated N maintains uncompacted viral RNA to promote the protein’s transcriptional function.