SARS-CoV-2 Nonstructural Protein 3 Remodels the Phosphorylation of Target Proteins via Protein-Protein Interactions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with a significant impact on human health. The molecular basis of its pathogenicity remains incompletely understood. The viral nucleocapsid (N) protein, the most abundant protein expressed during SARS-CoV-2 infection, is thought to contribute to disease progression. Yet, its interaction network in the context of viral infection remains largely unexplored. Here, we generated a recombinant (r)SARS-CoV-2 expressing a Strep-tagged N protein by using a reverse genetics system. Affinity purification and mass spectrometry identified an interaction between SARS-CoV-2 N protein and the nonstructural protein 3 (NSP3). Domain mapping revealed that the N dimerization domain and the N-terminal region of NSP3 mediate this interaction. Notably, an N protein mutant lacking its N-terminal domain exhibited enhanced binding to NSP3 and underwent dephosphorylation, implicating NSP3 as a potential viral phosphatase. We further found that NSP3 interacts with Interferon Regulatory Factor 3 (IRF3), a key transcription factor involved in host type I interferon (IFN-α/β) antiviral response. SARS-CoV-2 NSP3 expression suppressed poly(I:C)-induced IRF3 phosphorylation and broadly reduced cellular phosphorylation levels in a dose-dependent manner. These findings suggest that SARS-CoV-2 NSP3 modulates host phosphorylation dynamics to subvert antiviral signaling and facilitate viral replication.