Molecular Mechanism Of UBE2S Inhibition Of Type Ⅰ Interferon Production

The innate immunity system acts as the first line to defense against the invasion,replication and spread of pathogens and plays a key role in triggering adaptive immunity.Upon viral infection,the Pattern recognition receptors（PRRs）sense and recognize PAMPs,which activates innate immune response signal pathway,and ultimately promotes type I interferon（IFN）production and pro-inflammatory cytokines expression.Ubiquitination plays an indispensable role in almost every step in the innate immune signal cascade via ubiquitin enzymes（E1,E2 and E3）and deubiquitinatinases（DUBs）.They coordinate and accurately regulate the dynamic and reversible ubiquitination process,thereby participating in the regulation of host innate immune responses.Recently,a large number of E3 ubiquitin ligases have been identified and their roles in participating in the regulation of the innate immune response are widely investigated.However,there are few studies on ubiquitin-conjugating enzyme（E2）,therefore,the regulation effects of E2 on innate immune responses are still unkown.In order to identify which E2 ubiquitin-conjugating enzyme is involved in the innate immune signaling pathway,we first screened 35 members of ubiquitin-conjugating enzyme family with a dual luciferase reporter gene system,and found that the E2 ubiquitin-conjugating enzyme 2S（UBE2S）has a significant inhibitory effect on the production of IFN-β-induced by DNA-and RNA-viruses.To examine the role of UBE2S plays in type I IFN production,the function of UBE2S was first verified by overexpression experiments.We found that overexpression of UBE2S significantly inhibited the production of IFN-βupon Se V,VSV,EMCV and HSV-1 infection.Whereas knockout of UBE2S expression potentiated virus infection-induced IFN-βproduction,and inhibited EMCV replication.These results suggest that UBE2S is a negative regulator in the IFN-I signaling pathway.In order to further verify and explore the molelar mechanism（s）of UBE2S negatively regulating typeⅠIFN production,various molecular biology experimental techniques were performed.Subsequently,we found that UBE2S targets TBK1 to negatively regulate type I IFN production.Co-immunoprecipitation（Co-IP）and ubiquitination experiments proved that UBE2S inhibited the K63 ubiquitination of TBK1.However,we found the negative regulation of type I IFN production by UBE2S is independent on its E2or E3 enzyme activity.The Co-IP results revealed that UBE2S recruited USP15.We also noticed that UBE2S and USP15 synergistically inhibited the K63 ubiquitination of TBK1 upon virus infection.At the same time,we designed and generated UBE2S knockout cell line to prove that the inhibitory effect of USP15 on TBK1 ubiquitination depends on the expression of UBE2S.Taken together,in this study,we found that UBE2S recruits the deubiquitinating enzyme USP15 to inhibit the K63 ubiquitination of TBK1.UBE2S knockout mice were generated by Cre-Loxp and CRISPR/Cas9 technology.Ube2s^-/-mice and their WT littermates were challenged with either EMCV or HSV-1 by intraperitoneal injections.The m RNA levels of Ifnb1 in the heart and brain from Ube2s^-/-mice were significantly higher than those in other organs from WT mice after infection with EMCV.Correspondingly,the protein levels of IFN-βin serum from Ube2s^-/-mice were also significantly increased.Consistent with these results,the EMCV genomic RNA copy number and viral titers in the heart and brain were significantly lower in Ube2s^-/-mice than in WT mice.Moreover,Ube2s^-/-mice were more resistant to EMCV infection in challenge experiments.In contrast,the m RNA levels of Ifnb1 in the heart and brain from Ube2s transgenic mice were significantly lower than that of WT mice after infection with EMCV.Additionally,Ube2s transgenic mice results showed increased EMCV genomic RNA copy number in the heart and brain and decreased survival ability compared with WT mice.Based on these data,we concluded that UBE2S deficiency enhanced host antiviral immune responses,which resulted in the inhibition of both RNA virus and DNA virus replication and the reduction in virus infection-mediated pathological injury.In conclusion,we firstly proved that the UBE2S negatively regulates the production of type I interferon through in vivo and in vitro experiments.Our findings reveal the role of the UBE2S-USP15-TBK1 axis in regulating the host antiviral innate immune responses,enriching the regulatory network involved in the production of type I interferon by ubiquitination modification.In addition,these results increase‘our understanding of the function of UBE2S,providing an important reference for studying the functions of other E2 ubiquitin-conjugating enzyme family members.