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. IMPORTANCE: Understanding host-virus and virus-virus interactions is essential for uncovering mechanisms of viral replication and immune evasion, and for identifying targets for rational antiviral intervention. While previous screens using individually expressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins have revealed host factors involved in infection, they could not capture virus-virus protein interactions or virus-host interactions in the context of infection. Here, we engineered a recombinant (r)SARS-CoV-2 expressing a Strep-tagged nucleocapsid (N) protein to identify viral protein interactions with SARS-CoV-2 during active infection. We identified an interaction between the viral N protein and the nonstructural protein 3 (NSP3) and uncovered a previously unrecognized role for NSP3 in regulating viral and host protein phosphorylation, interaction with interferon regulatory factor 3 (IRF3), and regulation of innate immune response. This work highlights a powerful approach for dissecting protein interaction networks occurring during SARS-CoV-2 infection and suggests new targets for therapeutic development against SARS-CoV-2.