Journal of Molecular Biology, volume 433, issue 8, pages 166875

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

Dorothy E D P Hawkins 2
Jake L R Smith 2
Oliver W Bayfield 2
De-Sheng Ker 2
Oleg I. Klychnikov 3
A.A. Antson 2
Show full list: 9 authors
Publication typeJournal Article
Publication date2021-04-01
scimago Q1
SJR2.212
CiteScore11.3
Impact factor4.7
ISSN00222836, 10898638
Molecular Biology
Structural Biology
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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