Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation
- Creators
- Kubinski, Hannah C.
- Despres, Hannah W.
- Johnson, Bryan A.
- Schmidt, Madaline M.
- Jaffrani, Sara A.
- Mills, Margaret G.
- Lokugamage, Kumari
- Dumas, Caroline M.
- Shirley, David J.
- Estes, Leah K.
- Pekosz, Andrew
- Crothers, Jessica W.
- Roychoudhury, Pavitra
- Greninger, Alexander L.
- Jerome, Keith R.
- Martorelli Di Genova, Bruno
- Walker, David H.
- Ballif, Bryan A.
- Ladinsky, Mark S.
- Bjorkman, Pamela J.1
- Menachery, Vineet D.
- Bruce, Emily A.
Abstract
The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Acknowledgement
We would like to thank the University of Vermont Proteomics Core Facility, Drs Matthew Poynter, Dimitry Krementsov and Joyce Oetjen for technical assistance.
The authors thank the Caltech Beckman Institute CryoEM Facility for use and maintenance of the Tecnai T12 TEM.
Funding
This study was supported by an American Heart Association predoctoral fellowship (H.W.D.; 10.58275/AHA.23PRE1020524.pc.gr.161030), George Mason University Fast Grant (P.J.B.) NIH (NIAID) Award AI165075 (P.J.B.), NIH (NIAID) Award 1R01AI153602-01 (V.D.M.), Investigators in the Pathogenesis of Infectious Disease, Burroughs Wellcome Fund (V.D.M.) and NIH (NIGMS) award P30GM118228-04 (E.A.B) and UVM start-up funds (EAB).
Contributions
Conceptualization (HCK, HWD, EAB), Formal Analysis (HCK, HWD, BAJ, CMD, DJS, BAB, VDM, EAB), Investigation (HCK, HWD, BAJ, MMS, SJ, KL, CMD, DHW, BAB, MSL), Reagents (MGM, AP, JWC, PR, ALG, KRJ, VDM), Writing-Original Draft (HCK, HWD, EAB), Writing-Review & Editing (All authors), Visualization (HCK, HWD, BAJ, MSL, EAB), Supervision (BAB, PJB, VDM, EAB), Funding Acquisition (VDM, EAB).
Data Availability
All raw sequencing data are available in the NCBI Bioproject ID PRJNA1083584. Individual peptides identified in proteomics experiments are included in supplementary table 1, raw spectrometry files are available upon request.
Conflict of Interest
HCK, HWD, BAJ, VDM and EAB have filed a patent on the use of mutations in the nucleocapsid linker as a means of increasing nucleocapsid protein levels. V.DM. has filed a patent on the reverse genetics system and reporter SARS-CoV-2. Other authors declare no competing interests.
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Additional details
- PMCID
- PMC10979914
- Predoctoral Fellowship 10.58275/AHA.23PRE1020524.pc.gr.161030
- American Heart Association
- George Mason University
- AI165075
- National Institutes of Health
- 1R01AI153602-01
- National Institutes of Health
- Burroughs Wellcome Fund
- P30GM118228-04
- National Institutes of Health
- University of Vermont
- Caltech groups
- Division of Biology and Biological Engineering, Richard N. Merkin Institute for Translational Research