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

The coronavirus nucleocapsid protein, N, plays a crucial role in the viral lifecycle by being part of the Replication-Transcription Complex alongside Nsp3. Recent studies have revealed that varying levels of phosphorylation in N by host kinases regulate its functions during infection. However, little is known about the mechanisms controlling N dephosphorylation and its impact on the virus.

In this research, we discovered that SARS-CoV-2 Nsp3 acts as a central regulator of N dephosphorylation by recruiting the phosphatase PP1/{gamma} through a shared binding region in the Ubl1 domain. When this binding region is missing, N dephosphorylation is stopped, which in turn prevents the production of subgenomic RNA. We compared the interactions of phospho-mimicking N to those with phospho-deficient N, and further examined the role of SRSF1, a host splicing factor, which works with unphosphorylated N to boost subgenomic RNA transcription.

These findings reveal a new way in which Nsp3 helps N dephosphorylate through PP1/{gamma}, enabling coronavirus subgenomic RNA replication and providing a potential target for antiviral treatments.