Nsp3 Ubl1-orchestrated dephosphorylation of N protein promotes coronaviral subgenomic RNA synthesis

The coronavirus nucleocapsid (N) protein is indispensable for the viral lifecycle as part of the viral Replication-Transcription Complex together with Nsp3. Recent research demonstrated that phosphorylation of N by several host kinases intricately regulates its functions during infection. However, the mechanisms that control N dephosphorylation, and its physiological consequence, remain poorly understood. Here, we show that SARS-CoV-2 Nsp3 is a key orchestrator of N dephosphorylation by recruiting the phosphatase PP1α/γ via a conserved PP1-binding motif in the Ubl1 domain. Disruption of this motif abolishes N dephosphorylation and selectively impairs subgenomic RNA synthesis. Comparative interactome proteomics analysis of phospho-mimic vs. phospho-deficient N, together with functional validation, revealed that the host splicing factor SRSF1 cooperates with unphosphorylated N to promote subgenomic RNA transcription. These findings uncover an unrecognized mechanism in which Nsp3-guided N dephosphorylation by PP1α/γ enables coronavirus subgenomic RNA replication and highlight a potentially targetable axis for antiviral intervention.