IFI16 restricts SARS-CoV-2 replication by disrupting nucleocapsid-driven phase separation.

IFI16 is an interferon-inducible protein that senses viral DNA and can also restrict RNA virus replication. Here, using IFI16 knockout cellular models, we identify IFI16 as a host restriction factor that limits SARS-CoV-2 replication. Upon SARS-CoV-2 infection, IFI16 relocalizes from the nucleus to the cytoplasm, where it binds both the nucleocapsid protein and the viral genome. This impairs SARS-CoV-2 replication by inhibiting viral RNA-induced condensate formation of the nucleocapsid protein. Finally, we extend our analysis to other human coronaviruses and observe that IFI16 depletion also enhances OC43 replication, whereas it is associated with reduced NL63 replication, highlighting a differential, virus-specific effect of IFI16 on coronavirus infections. Overall, these findings provide mechanistic insights into how the absence of IFI16 creates a cellular environment that supports SARS-CoV-2 replication.