Direct interaction between human DDX1 and SARS-CoV-2 nucleocapsid protein is regulated by phosphorylation

The nucleocapsid (N) protein of SARS-CoV-2 is essential for viral replication and transcription, in part through interactions with host proteins. Here, we delineate distinct mechanisms underlying N protein association with human RNA helicases DDX1 and DDX21. Co-immunoprecipitation assays in HEK293 cells modified to express N protein revealed that DDX1 binding requires the N protein serine-arginine (SR) region, as SR deletion markedly reduced interaction. Inhibition of glycogen synthase kinase-3 (GSK-3), which targets the SR region, serine-to-alanine substitutions within the SR region, and alkaline phosphatase treatment of extract, respectively, demonstrated that phosphorylation of the SR region is critical for DDX1 binding. Furthermore, phosphorylated or phospho-mimetic SR peptides both prevented N protein-DDX1 complex formation and disrupted preformed complexes in vitro, whereas unphosphorylated peptides had no effect, confirming a phosphorylation-dependent binding mechanism. In contrast, interaction with DDX21 was unaffected by SR deletion or phosphorylation status and required both the N- and C-terminal domains of the N protein. RNase treatment enhanced N-DDX21 association without altering N-DDX1 interactions, indicating distinct regulation by RNA. Domain mapping of the two helicases identified the DDX1 N-terminal and the DDX21 C-terminal domains as interfaces that bind the N protein. Together, these findings support phosphorylation-dependent recruitment of DDX1 versus phosphorylation-independent engagement of DDX21, highlighting mechanistically distinct strategies by which SARS-CoV-2 N co-opts host helicases.