The Function And Molecular Mechanisms Of USP1-UAF1 Deubiquitinase Complex In Innate Antiviral Immune Responses

ObjectiveToll-like receptors(TLRs)and RIG-I-like receptors(RLRs)recognize pathogen-associated molecular patterns(PAMPs),and then recruit various adaptors to activate TANK-binding kinase 1(TBK1).Activated TBK1 then phosphorylates IFN regulatory factor 3(IRF3),triggers its dimerization and nuclear translocation,where it forms active transcriptional complexes that bind to IFN stimulation response elements(ISRE)and triggers type I IFN genes transcription.Type I IFN binds to I type Interferon receptor and promotes the production of numerous anti-viral genes through the JAK/STAT pathway.Therefore,TBK1 as an important kinase,its optimal activation is crucial for initiation of innate antiviral immunity and maintenance of immune homeostasis.Although several E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating its polyubiquitination,the functions of deubiquitinase on TBK1 activity remain largely unclear.Ubiquitin specific peptidase 1(USP1),a member of USP family,could deubiquitinate a wide range of substrates.The deubiquitinase activity of USP1 could be promoted by its binding partner USP1-associated factor 1.UAF1 and USP1 form a deubiquitinase complex,which participates in a variety of biological processes,such as regulation of DNA repair processes and tumor pathogenesis.However,the potential roles of USP1-UAF1 complex in immune system,especially in the innate antiviral immunity,remain unknown.Here,we found virus infection induced expression of USP1 and UAF1.we showed that USP1-UAF1 complex inhibited TLR3/4 and RIG-I induced IFN regulatory factor 3(IRF3)activation and subsequent IFN-? secretion.USP1 and UAF1 bound to TBK1,removed its K48-linked polyubiquitination,and then stabilized TBK1.Therefore,our results outline a novel mechanism for the control of TBK1 activity,and suggest USP1-UAF1 complex as a potential target for the prevention of viral diseases.Materials and Methods1.The effects of USP1-UAF1 on IFN-? production1.1 Mouse peritoneal macrophages were treated with increasing amount of ML323(0,15,30 60uM),a selective USP1-UAF1 inhibitor for 4 h and then infected with Sendai virus(SeV).ELISA analyzed IFN-? production.1.2Designed and synthesized the USP1,UAF1 specific siRNA.Mouse peritoneal macrophages were transfected with siRNA for 36h by Interferin,then simulated with LPS or infected with SeV.ELISA analyzed IFN-p,TNF-a and IL-6 production.1.3Builded USP1,UAF1 expression plasmids and cotransfected to RAW264.7 cells by JetPE1 for 24h,then analyzed luciferase activity of IFN-? or NF-?B reporter plasmid after stimulated with LPS,poly(I:C)or infected with SeV.1.AWestern blot analysis of UAF1 and USP1 expression in mouse peritoneal macrophages infected with SeV or stimulated with IFN-? for indicated time periods.2.USP1-UAF1 complex enhances IRF3 activation2.1HEK293T cells were cotransfected with USP1 or UAF1 expression plasmid,various receptor plasmids and IRF3 or NF-?B reporter plasmid,then analyzed luciferase activity of IRF3 or NF-?B reporter plasmid induced by different adaptors including TRIF,TBK1,MyD88,TRIF or RIG-I.2.2Mouse peritoneal macrophages were transfected with Ctrl siRNA,USP1 siRNA,UAF1 siRNA for 36h.Western blot analyzed the change of IRF3?STAT1 phosphorylation when stimulated with LPS or infected with SeV.3.USP1-UAF1 interacts with TBK13.1HEK293T cells were cotransfected with USP1 or UAF1 expression plasmid,various receptor expression plasmids and IFN-? reporter plasmid,then analyzed luciferase activity of IFN-P reporter plasmid inducted by RIG-I,MDA5,MAVS,TRIF,TBK1,IRF3-5D;HEK293T cells were cotransfected with USP1 or UAF1 expression plasmid,TBK1 expression plasmid and IRF3,ISRE,NF-?B reporter plasmids,then analyzed luciferase activity of IRF3,ISRE,NF-?B reporter plasmid induced by TBK1.3.2Western blot analysis of UAF1 and USP1 expression and translocation of USP1 in mouse peritoneal macrophages treated with LPS for indicated time periods.3.3HEK293T cells were cotransfected with USP1,UAF1 expression plasmid,various adaptor expression plasmids by Lipofectamine2000 for 24h,Lysates were subjected to immunoprecipitation.Western blot analyzed the interaction between USP1,UAF1 with different adaptors in TLR/RLR pathway.3.4Mouse peritoneal macrophages were stimulated with LPS for indicated time periods.Lysates were subjected to immunoprecipitation.Western blot analyzed the interaction between USP1,UAF1 with TBK1,IRF3 in vivo.4 USP1-UAF1 impacts ubiquitination and expression of TBK14.1HEK293T cells were cotransfected with TBK1,Ub or k48-Ub,USP1-WT,USP1-C90S or UAF1 expression plasmids by Lipofectamine2000 for 24h.Lysates were subjected to immunoprecipitation.Western blot analyzed the function of USP1,USP1-C90S,UAF1 in ubiquitination of TBK1.4.2HEK293T cells were transfected with different dose of USP1 or UAF1 expression plasmid by Lipofectamine2000 for 24h.TBK1 and IRF3 expression was analyzed by Western blot.4.3Mouse peritoneal macrophages were treated with ML323 for 4h,then stimulated with LPS.TBK1 expression was analyzed by Western blot.4.4Mouse peritoneal macrophages were transfected with Ctrl siRNA,USP1 siRNA,UAF1 siRNA by Interferin for 36h,then stimulated with LPS or infected with SeV.TBK1 expression was analyzed by Western blot.5 USP1-UAF1 complex regulated virus induced IFN-? in vitro and in vivo5.1 Mouse peritoneal macrophages were treated with ML323,ELISA analyzed virus induced IFN-?.RT-PCR analyzed IFN-? mRNA and downstream response gene mRNA and VSV RNA replicates in mouse peritoneal macrophages.5.2 C57BL/6 mice were intraperitoneally injected with thioglycolate to elicit peritoneal macrophages.After 3 days,the mice were treated with ML323 or DMSO,and then infected with VSV i.p.for 12 h.IFN-? expression in lavage was examined by ELISA.5.3 C57BL/6 mice(male,8weeks)were intraperitoneally injected with ML323(10mg/kg)for 6h,then injected with VSV(Vesicular Stomatitis Virus)8h.ELISA analyzed serum level of IFN-?.RT-PCR analyzed IFN-? mRNA and VSV RNA replicates in liver,lung and spleen.5.3 C57BL/6 mice were pretreated with ML323 or DMSO,and then infected with VSV i.p.for 18 h.H&E staining of lung tissue sections.Results1.USP1-UAF1 enhances IFN-P productionML323 greatly inhibited the secretion of IFN-? in mouse peritoneal macrophages infected with SeV in a does-dependent matter.USP1 and UAF1 knockdown reduced the secretion of IFN-? inducted by LPS,poly(I;C)and SeV in mouse peritoneal macrophages;USP1 and UAF1 overexpression significantly enhanced luciferase activity of IFN-P in RAW267.4 cells stimulated with LPS,poly(I;C),SeV.USP1 or UAF1 overexpression had no effect on luciferase activity of NF-?B.2.USP1-UAF1 enhances IRF3 activationOverexpression of UAF1 and USP1 both significantly enhanced TRIF and TBK1 induced IRF3 reporter activation.However,USP1 or UAF1 had no effect on MyD88,TRIF and RIG-I induced NF-?B activation.UAF1 and USP1 knockdown both markedly inhibited LPS and SeV induced phosphorylation of IRF3 and STAT1.3.USP1 and UAF1 interact with TBK1UAF1 and USP1 were both coprecipitated with TBK1.However,UAF1 and USP1 were not coprecipitated with IRF3.Next,endogenous interaction was examined in macrophages stimulated with LPS.UAF1 and USP1 interacted with TBK1 upon LPS stimulation.However,no interaction was observed between USP1/UAF1 and IRF3.In addition,USP12 and USP46 were not associated with TBK1.4.USP1-UAF1 complex removes K48-linked ubiquitination of TBK1 and stabilizes its expressionUAF1 and USP1 both markedly inhibited total and K48-linked ubiquitination of TBK1;UAF1 and USP1 overexpression both greatly enhanced TBK1 protein expression in a dose-dependent manner in HEK293T cells;UAF1 and USP1 knockdown also significantly decreased TBK1 protein level in mouse peritoneal macrophages.5.ML323 enhances innate antiviral responseML323 treatment significantly attenuated IFN-? and ISG-15 expression,and enhanced VSV replication in macrophages;ML323 treatment reduced IFN-? in serum and lavage,reduced IFN-? mRNA and had more virus replication in liver,lung and spleen.H&E staining of lung tissue sections showed more inflammatory in C57BL/6 mice treated by ML323.Conclusion1.USP1-UAF1 deubiquitinase complex specially bounds to TBK1 and removes its K48-linked polyubiquitination,and then inhibits the degradation process of TBK1 enhances TLR/RIG-I inducted IFN-? production.2.ML323,a selective USP1-UAF1 inhibitor,inhibits innate antiviral response and promotes virus replication in virus infection model.Innovation and Significance1.Revealing a novel mechanism for the control of TBK1 activity.2.Identifing USP1-UAF1 deubiquitinase complex as a potential target for the prevention of viral diseases.