Assembly of SARS-CoV-2 ribonucleosomes by truncated N∗ variant of the nucleocapsid protein

• Published in: The Journal of biological chemistry Vol. 299; no. 12; p. 105362
• Main Authors: Adly, Armin N., Bi, Maxine, Carlson, Christopher R., Syed, Abdullah M., Ciling, Alison, Doudna, Jennifer A., Cheng, Yifan, Morgan, David O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2023
• Subjects: COVID-19 - virology; Humans; N protein; nucleocapsid; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - metabolism; Nucleocapsid Proteins - ultrastructure; nucleosome; Phosphorylation; plus-stranded RNA virus; RNA binding protein; RNA virus; RNA, Viral - metabolism; RNA, Viral - ultrastructure; SARS-CoV-2; SARS-CoV-2 - genetics; SARS-CoV-2 - metabolism; SARS-CoV-2 - ultrastructure; SARS-CoV-2 variant; Virus Assembly - genetics; RTC; SARS-CoV-2 variant; N protein; RNA virus; RNA binding protein; EM; vRNP; ER; nucleocapsid; SARS-CoV-2; CTD; TRS; nucleosome; plus-stranded RNA virus; IDT; VLP; LH; phosphorylation; NTD; SR
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2023.105362

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N∗ or Δ(1–209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1–209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1–209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1–209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.