C-Cbl-mediated Ubiquitination Of IRF3to Negatively Regulate Antiviral Innate Immunity

TLR、RLR and NLR are very important receptors recogniting pathogenic microorganisms and initialing the innate immunity and adaptive immunity. The innate immune system constitutes the first line of host defense against viral infection. After recognizing invading viruses, pattern recognition receptors (PRRs) activate downstream signaling pathways to initiate immune response through the induction of I interferons (IFNs) to clear virus. Viral infection causes activation of transcription factor interferon regulatory factor (IRF)3, which is critical for production of IFN-P and innate antiviral immune response. Previous studies have demonstrated that ubiquitination and deubiquitination of the IRF3have emerged as critical regulatory processes in the virus-triggered type I IFN induction pathway.Casitas B-lineage lymphoma (c-Cbl) is a proto-oncogene with widespread mutations identified in hematopoietic malignancies. Acting mainly as an ubiquitin E3ligase, c-Cbl is a critical negative regulator of receptor tyrosine kinases (RTKs). The tyrosine kinase binding (TKB) domain of c-Cbl is required for its binding with RTKs, and the RING finger domain is essential for its ubiquitin E3ligase activity. In previous reports, c-Cbl plays an important role in antiviral innate immunity by regulated RIG-I and IRF8etc. In this study, we discovered that c-Cbl could down-regulate PRRs induced TEF-β production, we used specific siRNA to knock-down c-Cbl mRNA, the results demonstrated that c-Cbl decrease could promote production of IFN-β after VSV, poly(I:C) and LPS stimulation. Then we used c-Cbl to screen the key proteins in anti-virus signaling pathway, contain of RIG-I、MAVS、TBK1and IRF3by Luciferase reporter gene system, finally we confirm that c-Cbl can influence IRF3stabilization, but not in mRNA level. Furthermore, we confirm that IRF3could interact with c-Cbl by Immunoprecipitation. Immunoblot analysis of IRF3ubiquitination in293T cells cotransfected with IRF3、Ub、c-Cbl plasmids, we found that c-Cbl promoted IRF3K48-linked ubiquitination and enhanced IRP3degradation.In conclusion, we identified c-Cbl promoted K48-linked polyubiquitination and degradation of IRF3, which represents an important pathway in maintaining proper low amounts of type I IFN under physiological conditions, and for restraining its magnitude when the antiviral innate response intensifies. Thus, our study not only provides new insight into the molecular mechanisms for termination of excessive immune responses, but also provides a potential target for drug development against viral infection-related and autoimmune diseases.