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PreprintCited by 9514-3-3 protein interactionsSARS-CoV-2 and COVID-19 ResearchAnimal Virus Infections Studies

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The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

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Tugaeva, Kristina V., Hawkins, Dorothy E. D. P., Smith, Jake L. R., Bayfield, Oliver W., Ker, De-Sheng, Sysoev, Andrey A., Klychnikov, Oleg I., Antson, Alfred A., Sluchanko, Nikolai N.·2020·Journal of Molecular Biology
DOI 10.1101/2020.12.26.424450PMC PMC7863765

Preprint — published as 10.1016/j.jmb.2021.166875 · Paper page

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Abstract

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent K D to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention. Highlights: SARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

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Metadata

Authors
Tugaeva, K. V., Hawkins, D. E. D. P., Smith, J. L. R., Bayfield, O. W., Ker, D.-S., Sysoev, A. A., Klychnikov, O. I., Antson, A. A., & Sluchanko, N. N.
Affiliations
  • A N Bach Institute of Biochemistry (RU)
  • University of York (GB)
  • Lomonosov Moscow State University (RU)
  • Russian Academy of Sciences (RU)
Corresponding author
  • Antson, Alfred A.
  • Sluchanko, Nikolai N.
ORCID
Venue
Journal of Molecular Biology
Publisher
openRxiv
Published
2020-12-27
Volume
433
Issue
8
Pages
166875-166875
ISSN
0022-2836 (ISSN-L)
DOI
10.1101/2020.12.26.424450
PMC
PMC7863765
OpenAlex
W3128571690
Cited by
95
References
1
Citations by year
2026: 7 · 2025: 15 · 2024: 19 · 2023: 16 · 2022: 24 · 2021: 14
FWCI
5.70
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Molecular Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (submittedVersion)
Funders
  • Wellcome Trust (206377, 206377/Z/17/Z)
  • Russian Science Foundation (19‐74‐10031)
  • Ministry of Science and Higher Education of the Russian Federation
  • Biotechnology and Biological Sciences Research Council (BB/R506412/1)
  • Russian Academy of Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › 14-3-3 protein interactions
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
Keywords
SARS-CoV-2 nucleocapsid protein, 14-3-3 proteins, protein phosphorylation, host-pathogen interaction, protein-protein binding, nucleocytoplasmic shuttling, protein kinase A, binding affinity, therapeutic target, zoonotic coronaviruses
MeSH terms
Coronavirus Nucleocapsid Proteins, Amino Acid Sequence, Binding Sites, Escherichia coli, Humans, Mutation, Phosphopeptides, Phosphoproteins, Phosphorylation, Phosphoserine, Protein Binding, RNA, Viral, Substrate Specificity, Cyclic AMP-Dependent Protein Kinases, Protein Isoforms, 14-3-3 Proteins

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