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Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Corinne A. Lutomski, Tarick J. El-Baba, Jani R. Bolla, Carol V. Robinson
doi: https://doi.org/10.1101/2020.10.06.328112
Corinne A. Lutomski
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, OX13QZ Oxford, UK
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Tarick J. El-Baba
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, OX13QZ Oxford, UK
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Jani R. Bolla
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, OX13QZ Oxford, UK
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Carol V. Robinson
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, OX13QZ Oxford, UK
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  • For correspondence: carol.robinson@chem.ox.ac.uk
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Abstract

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

Competing Interest Statement

The authors have declared no competing interest.

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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.
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Posted December 28, 2020.
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Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response
Corinne A. Lutomski, Tarick J. El-Baba, Jani R. Bolla, Carol V. Robinson
bioRxiv 2020.10.06.328112; doi: https://doi.org/10.1101/2020.10.06.328112
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Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response
Corinne A. Lutomski, Tarick J. El-Baba, Jani R. Bolla, Carol V. Robinson
bioRxiv 2020.10.06.328112; doi: https://doi.org/10.1101/2020.10.06.328112

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