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

Armin N Adly; Maxine Bi; Christopher R Carlson; Abdullah M Syed; Alison Ciling; Jennifer A Doudna; Yifan Cheng; David O Morgan

doi:10.1016/j.jbc.2023.105362

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

J Biol Chem. 2023 Dec;299(12):105362. doi: 10.1016/j.jbc.2023.105362. Epub 2023 Oct 19.

Authors

Armin N Adly¹, Maxine Bi², Christopher R Carlson¹, Abdullah M Syed³, Alison Ciling³, Jennifer A Doudna⁴, Yifan Cheng⁵, David O Morgan⁶

Affiliations

¹ Department of Physiology, University of California, San Francisco, California, USA.
² Department of Biochemistry & Biophysics, University of California, San Francisco, California, USA.
³ J. David Gladstone Institutes, San Francisco, California, USA.
⁴ J. David Gladstone Institutes, San Francisco, California, USA; Department of Molecular and Cell Biology, University of California, Berkeley, California, USA; Howard Hughes Medical Institute, University of California, Berkeley, California, USA; Innovative Genomics Institute, University of California, Berkeley, California, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, California, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
⁵ Department of Biochemistry & Biophysics, University of California, San Francisco, California, USA; Howard Hughes Medical Institute, University of California, San Francisco, California, USA.
⁶ Department of Physiology, University of California, San Francisco, California, USA. Electronic address: David.Morgan@ucsf.edu.

Abstract

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.

Keywords: N protein; RNA binding protein; RNA virus; SARS-CoV-2; SARS-CoV-2 variant; nucleocapsid; nucleosome; phosphorylation; plus-stranded RNA virus.

MeSH terms

COVID-19 / virology
Humans
Nucleocapsid Proteins* / genetics
Nucleocapsid Proteins* / metabolism
Nucleocapsid Proteins* / ultrastructure
Phosphorylation
RNA, Viral / metabolism
RNA, Viral / ultrastructure
SARS-CoV-2* / genetics
SARS-CoV-2* / metabolism
SARS-CoV-2* / ultrastructure
Virus Assembly / genetics

Substances

Nucleocapsid Proteins
RNA, Viral
nucleocapsid phosphoprotein, SARS-CoV-2

Abstract

MeSH terms

Substances

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