The Effect And Molecular Mechanism Of TRIM50 In Antiviral Immunity

PurposesInnate immunity is the first line of defense against viral infection.The mechanism of activation and regulation of immune response is precise and complex.Tripartite motif(TRIM)proteins play an important role in the regulation of innate immune signaling pathways.TRIM50,a new member of TRIM protein family,has been reported to play an important role in tumor therapy and regulation of autophagy,but the role of TRIM50 in innate immunity remains to be clarified.In this study,we aim to explore the role and molecular mechanism of TRIM50 in the regulation of antiviral innate immune response,and we try to define new mechanisms of antiviral immune regulation,and provide new strategies for the treatment of viral infection related diseases in clinical practice.MethodsTo explore the role of TRIM50 in antiviral immune response,peritoneal macrophages were isolated from WT and Trim50-/-mice,and further infected with Herpes simplex virus(HSV)and Vesicular stomatitis virus(VSV).The effect of TRIM50 on the antiviral immune response of mouse macrophages was detected by qPCR,ELISA,western blot and viral plaque assay.Dual-luciferase reporter gene assay and co-immunoprecipitation were used to screen the molecules that interacted with TRIM50 in the antiviral immune pathway,and immunofluorescence microscopy was applied was used to analyze their co-localization in the cells.The mechanism of the regulation of target molecules by TRIM50 was further explored by immunoprecipitation and ubiquitination analysis.The viral infection mouse model was constructed to detect the effect of TRIM50 on the antiviral immune response in vivo.Results1.TRIM50 negatively regulates the antiviral immune responseThe peritoneal macrophages from WT and Trim50-/-mice were infected with HSV and VSV,and our data revealed that Trim50-/-macrophages produced higher levels of IFN-β,and higher levels of P-TBK1 and P-IRF3 proteins in the antiviral signaling pathway,indicating a stronger antiviral capability of Trim50-/-macrophages.These results indicated that TRIM50 played a negative regulatory role in antiviral immune response.2.TRIM50 interacts with TBK1The data of dual luciferase reporter gene assay showed that TRIM50 negatively regulated the antiviral immune response mediated by RIG-I and cGAS.Co-immunoprecipitation screening and immunofluorescence analysis showed that TRIM50 could interact with TBK1,a key molecule in the antiviral pathway,and the interaction gradually increased during the process of virus infection.3.TRIM50 removes K63 ubiquitination of TBK1 by recruiting BRCC3Mechanistically,TRIM50 negatively regulated the antiviral immune response by reducing the K63 ubiquitination level of TBK1.The data of target screening and co-immunoprecipitation verification showed that TRIM50 formed a complex with BRCC3 to negatively regulate K63 ubiquitination of TBK1.Knockdown of BRCC3 reversed the inhibitory effect of TRIM50 on the K63 ubiquitination of TBK1,suggesting that TRIM50 mediated K63-deubiquitination of TBK1 through BRCC3.The ubiquitination analysis of the BRCC3 enzyme activity dead mutant showed that BRCC3 mediated K63-deubiquitination of TBK1 through its deubiquitinating enzyme activity.4.TRIM50 negatively regulates antiviral immune response by recruiting BRCC3Brcc3 SiRNA was used to construct a BRCC3 knockdown bone marrow-derived macrophages(BMDM)cellular model.The data showed that after virus infection,the level of IFN-β in the BRCC3 knockdown group was significantly upregulated compared with the control group,indicating that BRCC3 knockdown enhanced the antiviral immune response.The BMDM cellular model with simultaneous knockdown of TRIM50 and BRCC3 was constructed.The data showed that TRIM50 played a negative regulatory role in antiviral immune response through the deubiquitination effect of BRCC3.5.TRIM50 negatively regulated the antiviral immune response in vivo.WT and Trim50-/-mice were infected by VSV to construct viral infection models.The levels of IFN-β and viral load in the serum and organs of mice were detected.The data showed that the level of IFN-β in the serum and organs of Trim50-/-mice was higher,the virus titer was lower,and the survival time was longer,indicating that TRIM50 negatively regulated the antiviral immune response in vivo.Conclusions and innovations1.TRIM50 plays a negative regulatory role in antiviral immunity.2.TRIM50 interacted with TBK1.TRIM50 inhibited the K63 linked ubiquitination modification of TBK1 the and negatively regulated the activation level of TBK1.3.TRIM50 negatively regulates the antiviral immune response by recruiting BRCC3 to mediate K63-deubiquitination of TBK1.