The Functions And Mechanisms Of RNF115 In Promoting Cellular Antiviral Responses By Ubiquitinating MITA

The innate immune system deploys a variety of pattern recognition receptors(PRRs)to detect invading pathogens.Viral nucleic acids,including DNA and RNA,are a major class of pathogen-associated molecular patterns(PAMPs)that are recognized by PRRs and trigger robust innate immune responses.Viral DNA binds to and activates the DNA sensor cGAS,active c GAS then converts GTP and ATP into2'3'-cGAMP which further binds to MITA,an endoplasmic reticulum(ER)-localized adaptor protein.MITA subsequently undergoes oligomerization and migration and ultimately activates downstream transcription factors IRF3 and NF-kB to induce production of type I interferons(IFNs)and pro-inflammatory cytokines.As one of the most common post-translational modifications,ubiquitination is widely involved in a variety of biological processes.The activity and stability of MITA are strictly regulated by ubiquitination as well.It has been reported that K63-linked ubiquitination of MITA is crutial for its activation.Several studies have shown that TRIM32,TRIM56 and MUL1 mediate K63-linked ubiquitination of MITA.However,results from gene deletion studies suggest that knockout of any of them in mice had minimal effects on the ubiquitination of MITA after viral infection,indicating that the E3s responsible for K63-linked ubiquitination of MITA at the physiological level are still unclear and the underlying molecular mechanism and physiological significance await for further elucidation.In this study,we made?200 human E3s ligases expression vectors and performed an unbiased screening for MITA-interacted E3 ligases by transient transfection and co-immunoprecipitation(co-IP).This effort led to the identification of RNF115 as a MITA-interacting E3 ligase.We found that RNF115 interacted with endogenous MITA after HSV-1 infection.Knockdown or knockout of RNF115inhibits HSV-1-or cytoplasmic DNA-triggered expression of type I IFNs and proinflammatory cytokines and promotes HSV-1 replication.Rnf115^-/-mice were more susceptible to HSV-1 infection than the wild-type counterparts.The production of type I IFNs and proinflammatory cytokines in sera was impaired and the replication of HSV-1 was augmented in brains and spleens from Rnf115^-/-mice compared with Rnf115^+/+mice.Mechanistically,RNF115 interacted with and mediated K63-linked ubiquitination of MITA on K20,K224 and K289 residues after HSV-1 infection and promotes MITA oligomerization,migration and recruiment of downstream kinase TBK1,thereby promoting HSV-1-triggered antiviral responses.In addition,we reconstituted wild-type MITA and MITA(3KR)into Mita^-/-MLFs followed by infection with HSV-1.Results from immunoblot and q RT-PCR assays showed that downstream antiviral signaling was substantially impaired when reconstituted with MITA(3KR)compared to wild-type MITA.Overall,this study suggests a crucial role for RNF115 in promoting MITA activity and positively regulating innate antiviral signaling.