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The Mechanism of SARS-CoV-2 Nucleocapsid Protein Recognition by the Human 14-3-3 Proteins

Authors
  • Tugaeva, Kristina V.1
  • Hawkins, Dorothy E.D.P.2
  • Smith, Jake L.R.2
  • Bayfield, Oliver W.2
  • Ker, De-Sheng2
  • Sysoev, Andrey A.1
  • Klychnikov, Oleg I.3
  • Antson, Alfred A.2
  • Sluchanko, Nikolai N.1
  • 1 A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia
  • 2 York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom
  • 3 Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
Type
Published Article
Journal
Journal of Molecular Biology
Publisher
Elsevier
Publication Date
Apr 16, 2021
Volume
433
Issue
8
Identifiers
DOI: 10.1016/j.jmb.2021.166875
PMID: 33556408
PMCID: PMC7863765
Source
PubMed Central
Keywords
Disciplines
  • Fast Track
License
Unknown

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 KD 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 could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

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