Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host innate immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, DUBs that regulate this pathway have not been identified. Using a ubiquitin C-terminal electrophile, we profiled DUBs that function during influenza A virus (IAV) infection, and isolated OTUB1 as a key regulator of RIG-I dependent antiviral responses. OTUB1 was interferon-inducible, and interacted with RIG-I, viral PB2 and NS1. Upon infection, OTUB1 relocalised from the nucleus to mitochondrial membranes, and activated the RIG-I signalling complex via hydrolysis of K48 polyubiquitin chains and by forming a repressive complex with UBCH5c. Using a reconstituted system composed of in vitro translated [35S]IRF3, purified RIG-I, mitochondrial membranes and cytosol expressing OTUB1 variants, we recapitulated the mechanism of OTUB1-dependent RIG-I activation. A wide range of IAV NS1 proteins triggered proteasomal degradation of OTUB1, thereby antagonizing the RIG-I signalling cascade and antiviral responses.

OTUB1 is a key regulator of RIG-I dependent immune signalling and is targeted for proteasomal degradation by influenza A NS1