Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

Huaying Zhao; Abdullah M Syed; Mir M Khalid; Ai Nguyen; Alison Ciling; Di Wu; Wai-Ming Yau; Sanjana Srinivasan; Dominic Esposito; Jennifer A Doudna; Grzegorz Piszczek; Melanie Ott; Peter Schuck

doi:10.1093/nar/gkae256

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid

Nucleic Acids Res. 2024 Apr 8:gkae256. doi: 10.1093/nar/gkae256. Online ahead of print.

Authors

Huaying Zhao¹, Abdullah M Syed^{2

3}, Mir M Khalid², Ai Nguyen¹, Alison Ciling^{2

3}, Di Wu⁴, Wai-Ming Yau⁵, Sanjana Srinivasan¹, Dominic Esposito⁶, Jennifer A Doudna^{2

7

8

9

10

11}, Grzegorz Piszczek⁴, Melanie Ott^{2

12

13}, Peter Schuck^{1

14}

Affiliations

¹ Laboratory of Dynamics of Macromolecular Assembly, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
² Gladstone Institutes, San Francisco, CA 94158, USA.
³ Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA.
⁴ Biophysics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
⁵ Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
⁶ Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
⁸ HHMI, University of California, Berkeley, CA 94720, USA.
⁹ Department of Chemistry, University of California, Berkeley, CA 94720, USA.
¹⁰ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
¹¹ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, USA.
¹² Department of Medicine, University of California, San Francisco, CA 94143, USA.
¹³ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
¹⁴ Center for Biomedical Engineering Technology Acceleration, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.

PMID: 38587193
DOI: 10.1093/nar/gkae256

Abstract

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Published by Oxford University Press on behalf of Nucleic Acids Research 2024.

Grants and funding

ZIA EB000095-04/NH/NIH HHS/United States