Mechanisms Of BRCC36-mediated Regulation On STAT1 Stability And Its Innate Antiviral Immune Response

Objective:STAT1 is the first member of Signal Transducers and Transcription Activators family,and STAT1 plays an important role in host defense against viral infection.The role of STAT1 ubiquitination in host defense against viral infection and its mechanism remains unclear.This study focused on the role of deubiquitinase BRCC36 in the innate antiviral immune response.Methods:(1)Cells were transfected with BRCC36 overexpression plasmid or BRCC36 knockdown plasmid or BRCC36-QSQ(DUB activity-deficient mutant)plasmid and then infected with VSV-GFP or SeV or CVB3 or H1N1 viruses.The innate antiviral immune response of deubiquitinase BRCC36 was determined by using inverted fluorescence microscopy or Realtime qPCR or Western Blot.(2)Cells were transfected with BRCC36 overexpression plasmid or BRCC36 knockdown plasmid or BRCC36-QSQ(DUB activity-deficient mutant).Then the protein level of STAT1 and the ubiquitination of STAT1 was evaluated by Immunoprecipitation and Western Blot.(3)The DUB library was used to transfect cells,and the DUB interacting with both BRCC36 and STAT1 was screened by immunoprecipitation and Western Blot.The protein level of STAT1 and the ubiquitination of STAT1 regulated by the screened DUB were investigated by immunoprecipitation and Western Blot.Meanwhile,the effect of E3 ligase Smurfl on STAT1 protein level and ubiquitination was also evaluated by Immunoprecipitation and Western Blot.(4)Mapping:Construction of BRCC36 deletion mutants;Mutants of BRCC36 were transfected into cells,then the interaction of BRCC36 deletion mutants with Smurfl and USP13 was analyzed by immunoprecipitation and Western Blot.(5)Cells were infected with SeV viruses,and the correlation between cellular BRCC36 and STAT1 protein was explored by Western Blot.STAT1 protein stability in virus-infected cells regulated by BRCC36 was assessed by Western Blot.(6)In vivo experiments:Construction of mouse Flag-mBRCC3 plasmid.Flag-mBRCC3 overexpression plasmid was injected into the tail vein of mice by HGT(high pressure hydrodynamic),and Flag-mBRCC3 proteins were highly expressed in mice.The mice were anesthetized by intraperitoneal injection of pentobarbital sodium and infected with VSV-GFP virus,and finally the survival curve of mice was observed and recorded.The liver,kidney and lung were collected 14 days after infections,fixed in 4%formalin and then stained with hematoxylin-eosin.The liver,kidney and lung were collected at 3 days after virus infection,and then the VSV viral load was measured by Realtime qPCR.To investigate whether mBRCC3-mediated regulation on innate antiviral immune response in mice is dependent on STAT1,we treated hepatocytes isolated from control mice and Flag-mBRCC3-overexpressing mice with STAT1 activity inhibitor Fludarabine.Then cells were infected with VSV-GFP virus.Realtime qPCR was used to detect VSV viral load in hepatocytes.Results:(1)Inverted fluorescence microscopy or Realtime qPCR or Western Blot results showed that the deubiquitinase BRCC36 promotes the innate antiviral immune response,which is independent of its DUB activity.(2)The deubiquitinase BRCC36 physically interacts with STAT1,up-regulates STAT1 protein levels and deubiquitinates STAT1,which is independent of the DUB activity of BRCC36.BRCC36 deubiquitinates K63-linked ubiquitination of STAT1.(3)Deubiquitinase USP13 interacts with BRCC36 and STAT1.Deubiquitinase USP13 up-regulates STAT1 protein levels,and deubiquitinates K48-linked and K63-linked ubiquitination of STAT1.E3 ubiquitin ligase Smurfl ubiquitinates K48-linked ubiquitination of STAT1,and degrades STAT1 protein.(4)Mapping:Deubiquitinase USP13 is bound to N-terminus of BRCC36 protein and E3 ubiquitin ligase Smurfl is bound to the C-terminus of BRCC36 protein.USP13 and Smurfl bind to different regions of BRCC36 protein,respectively.(5)In SeV-infected cells,there is a positive correlation between cellular BRCC36 protein and STAT1 protein;Deubiquitinase BRCC36 promotes STAT1 protein stability in SeV-infected cells.(6)The mouse Flag-BRCC3 plasmid was successfully constructed and expressed in cells.Mouse Flag-BRCC3 injected into mice by HGT was successfully expressed in liver and kidney.However,mouse Flag-BRCC3 proteins are hardly to be detected in lungs.Mouse Flag-BRCC3 significantly inhibited virus load of VSV-GFP in the liver and kidney of mice.Mouse Flag-BRCC3 significantly promoted STAT1-mediated innate antiviral response and attenuated the pathological damage of liver and kidney caused by viral infection.Fludarabine is an inhibitor of STAT1 activity.BRCC3 can not inhibit virus load of VSV-GFP in mouse hepatocytes when treated with Fludarabine.Conclusion:The deubiquitinase BRCC36 regulates STAT1 ubiquitination by balancing the deubiquitinase USP13 and the E3 ubiquitin ligase Smurfl,thus stabilizing STAT1 protein levels,and finally promoting innate antiviral immune response.