Study On Anti - Virus Natural Immune Response And Mechanism Of RNF122 Negative Regulation

Upon virus infection, host cells detect viral nucleic acids and initiate antiviral innate immune responses by producing type I interferons (IFNs) and proinflammatory cytokines. As a cytoplasmic innate sensor for viral RNA, RIG-I undergoes a conformational change after binding of pathogenic RNA to the helicase domain and is recruited to the mitochondrial antiviral signaling (MAVS) adaptor. Downstream from MAVS, transcription factors IRF3 and NF-κB are activated to stimulate the expression of type I IFN and proinflammatory cytokines for host defense. The activation of RIG-I must be tightly regulated to remove pathogens effectively and prevent excessive inflammatory reactions to avoid harmful immunopathology. How to timely terminate RIG-I signaling to prevent overproduction of type I IFN and avoid inflammatory autoimmune disease need to be investigated.Post-translational modifications, particularly ubiquitination, are crucial for regulation of RIG-I activity. E3 ubiquitin ligases have been reported to be involved in the regulation of RIG-I signaling. But the previously unknown negative regulators of RIG-I signaling remain to be further identified.Here we report that RING finger protein 122 (RNF122), an E3 ubiquitin ligase, interacts with mouse RIG-I as demonstrated through screening the RIG-I-interacting proteins in RNA virus-infected cells using mass spectrometry. RNF122 contains a transmembrane (TM) domain in N terminus and RING finger domain in C terminus, suggesting its potential E3 ubiquitin ligase activity. Mouse RNF122 protein shares 97% homology with the human counterpart. RNF122 is widely expressed in many mouse tissues, with particularly abundant in immune organs and cells. Our studies demonstrate the colocalization of RNF122 and RIG-I with or without VSV infection in the cytoplasm of mouse peritoneal macrophages. Further, the transmembrane (TM) domain of RNF122 directly associates with the caspase activation and recruitment domains (CARDs) of RIG-I; this interaction effectively triggers RING finger domain of RNF122 to deliver the Lys-48-linked ubiquitin to the Lys115 and Lys146 residue of RIG-I CARDs and promote RIG-I degradation, resulting in a marked inhibition of RIG-I downstream signaling.Deficiency or knockdown of RNF122 significantly increased mRNA expression of IFN-β in macrophages upon infection with RNA virus (VSV and SeV) and transfection of poly(I:C). Furthermore, RNF122-/- macrophages secreted much more type I IFNs (IFN-α and IFN-β), TNF-α and IL-6 than RNF122+/+ macrophages in response to infection with RNA virus but not DNA virus. Deficiency or knockdown of RNF122 significantly increased the levels of p-IRF3 and p-p65 upon VSV infection in Immunoblot analysis.Collectively, these data demonstrate that RNF122 is a selective, negative regulator of RIG-I signaling cascade and can significantly suppress the antiviral innate response against RNA viral infection.Deficiency of RNF122 shows no defects in myeloid development and macrophage differentiation. But, RNF122-/- mice exhibited more production of type I IFNs and proinflammatory cytokines in response to RNA virus infection, resulting in less infiltration of inflammatory cells in the lungs and longer survival after lethal RNA virus infection than control groups. As a self-ubiquitination E3, RNF122 protein expression is increased in the infected cells and mice, which is important for restraining type I IFN overproduction.In conclusion, we identified RNF122 as a RIG-I-interacting protein which suppressed type I IFNs production by promoting K48-linked ubiquitination and degradation of RIG-I. The discovery that RNF122 is a negative regulator in the sensing of RNA virus provides new insight into the mechanism of regulation of cellular antiviral innate response and inflammation.