Graphical abstract 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 K D 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. 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 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.
【저자키워드】 Phosphorylation, host-pathogen interactions, protein–protein complex, nucleocytoplasmic shuttling, stoichiometry, 【초록키워드】 SARS-CoV-2, coronavirus, Infection, molecular mechanism, nucleocapsid protein, Protein, Control, SARS-CoV-2 N protein, mutant, Critical, function, affinity, Bacterial, association, Interaction, regulate, viral genome, Efficiency, cytoplasm, Abstract, tissue, therapeutic intervention, SARS-CoV-2 nucleocapsid, cellular pathway, isoforms, isoform, Host, regions, co-expression, Seven, bind, selected, conserved, reported, expressed, SARS-CoV N, phosphorylated, dimeric, monomeric, phosphorylation site, protein kinase A, zoonotic coronavirus, 【제목키워드】 SARS-CoV-2, Human, Recognition,