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

Zhao, H.; Syed, A. M.; Khalid, M. M.; Nguyen, A.; Ciling, A.; Wu, D.; Yau, W.-M.; Srinivasan, S.; Esposito, D.; Doudna, J. A.; Piszczek, G.; Ott, M.; Schuck, P.

doi:10.1101/2023.11.22.568361

bioRxiv · 10.1101/2023.11.22.568361

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

Zhao, H.·Syed, A. M.·Khalid, M. M.·Nguyen, A.·Ciling, A.·Wu, D.·Yau, W.-M.·Srinivasan, S.·Esposito, D.·Doudna, J. A.·Piszczek, G.·Ott, M.·Schuck, P.

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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 mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins 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 multi-valent 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.

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Zhao, H., Syed, A. M., Khalid, M. M., Nguyen, A., Ciling, A., Wu, D., Yau, W.-M., Srinivasan, S., Esposito, D., Doudna, J. A., Piszczek, G., Ott, M., Schuck, P.. 2023-11-23. Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid. https://doi.org/10.1101/2023.11.22.568361

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