1 × 10^6 mouse peritoneal macrophages, 2 × 10^5 HEK-293T or 2 × 10^5 RAW 264.7 cells were seeded into each well of 12-well plates, incubated overnight, and transfected with siRNA using INTERFERin, according to the manufacturer's instructions. 2 × 10^5 RAW 264.7 or HEK-293T cells were seeded into each well of 12-well plates or 5 × 10^5 293T cells were seeded into each well of 6-well plates, incubated overnight and transfected with DNA using jetPEI according to the manufacturer's instructions.

Induction of type I interferon is a fundamental cellular response to viral infection. Interferon regulatory factor 3 (IRF3) plays an essential role in Toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I) mediated induction of type I interferon and host antiviral responses. However, posttranslational regulation of IRF3 remains to be fully understood. In this study, we identified E3 ubiquitin ligase Casitas B-lineage lymphoma (c-Cbl) as a negative regulator for IRF3 protein stability and IFN-beta signal pathway. Knockdown of c-Cbl expression by small interfering RNA enhanced virus-induced IFN-beta production as well as cellular antiviral response, whereas overexpression of c-Cbl inhibited virus-induced IFN-beta signaling. Coimmunoprecipitation experiments demonstrated that c-Cbl interacted with IRF3 via TKB domain of c-Cbl and IRF association domain of IRF3, promoting K48-linked polyubiquitination and proteasomal degradation of IRF3. Therefore, our findings suggest that c-Cbl negatively regulates IFN-beta signaling and cellular antiviral response by promoting IRF3 ubiquitination and degradation, providing a new mechanism for control of type I interferon induction.