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-/{beta}) 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. SIGNIFICANCEUnderstanding virus-host and virus-virus interactions is essential for elucidating the mechanisms of viral replication and immune evasion. Previous studies using individually expressed SARS-CoV-2 proteins have identified host interacting factors but have largely overlooked interactions between viral proteins. Here, we engineered a recombinant SARS-CoV-2 virus expressing Strep-tagged nucleocapsid (N) protein, allowing the identification of both viral and host proteins interacting with N during live infection. We discovered an interaction between N and nonstructural protein 3 (NSP3), revealing a previously unrecognized role for NSP3 in modulating protein phosphorylation, interacting with Interferon Regulatory Factor 3 (IRF3), and regulating the innate immune response. This work demonstrates a powerful strategy for dissecting protein interaction networks during SARS-CoV-2 infection and identifies potential targets for therapeutic intervention.