SARS-CoV-2 nonstructural protein 3 remodels the phosphorylation of target proteins via protein-protein interactions

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.