The Role Of Deubiquitinase BAP1 In Antiviral Innate Immune Response And The Underlying Mechanisms

As the very first line of host defense against invading pathogens,innate immune system recognizes the highly conserved pathogen associated molecular pattern(PAMP)or explores the host derived danger associated molecular pattern(DAMP)via a series of pattern recognition receptors(PRRs)to monitor the pathogen infection or perceive the cell survival stress.The activation of PRRs will activate the relative downstream signaling pathways,which will eventually activate such transcriptional factors as NF-?B,AP-1 and IRF to drive the transcriptional activation of inflammatory cytokines,chemokines and interferon,to eliminate invading pathogens or abnormal host cells,and to protect the host.Post-translational modification(PTM)is an important mode of protein function regulation.Ubiquitination is co-regulated by the E1-E2-E3 ubiquitinase system and deubiquitinating enzymes,and plays important roles in many immune processes,such as antigen presentation,cell differentiation,immune defense and inflammation.Ubiquitin is a highly conserved small molecule polypeptide consisting of 76 amino acid residues with a molecular weight of 8.6 kDa.It can form a polyubiquitin chain through its own seven lysine residues—Lys 6,11,27,29,33,48,63,or form linear ubiquitination by head-to-tail connection between two ubiquitin.K48-linked ubiquitination had been extensively studied for the role in proteasome degradation,while the K63-linked ubiquitination had been reported to function in protein kinase activation.Deubiquitinating enzymes can regulate the ubiquitination on a certain protein site together with the ubiquitin modification system.Since ubiquitin can be linked in various ways,it can function widely and importantly in protein function regulation.Deubiquitinating enzymes belong to the superfamily of proteases and consist of cysteine proteases and metallo proteases.However,the function and mechanism of deubiquitination in innate immunity need to be further elucidated,and the role of deubiquitinating enzymes in innate immune response remains to be fully understood.In this study,we utilized the small interfering RNA(siRNA)library for the whole deubiquitinating enzyme family and intended to screen interest molecules with potent ability to regulate antiviral innate immune response.We screened a series of potential genes that regulate antiviral innate immune signaling in response to infection with VSV,SeV and HSV-1,respectively.Some of them have been reported to be consistent with the effects we screened,which confirmed the feasibility of our screening system.Among the screened deubiquitinating enzyme genes which have not been reported to function in innate immune regulation,we preferred BRCA1-associated protein 1(BAP1)as the regulatory effects are robust and consistent among different viral infection models,hoping to discover a general mechanism for the regulation of antiviral innate immune response.Studies have shown that BAP1 is involved in many important physiological processes,such as cell cycle,differentiation,transcription and DNA damage repair,however,the mutation of BAP1 can increase the incidence of malignant tumors,such as melanoma,mesothelioma and renal cell carcinoma.However,the role of BAP1 in antiviral innate immune response remains unclear.With functional experiments in vitro,we found that BAP1 could significantly increase the expression and secretion of type I interferons,which were induced by viral infection and transfection of exogenous nucleic acid mimics.This could enhance the ability of eliminating viral infection and inhibiting intracellular viral replication.Because the genetic defect of BAP1 is mouse embryos lethal,we attempted to construct a BAP1 lung-specific overexpression or expression silent mouse model by using lentivirus to infect the lungs of wild-type mice.Through the model of influenza virus infection in vivo,we discovered that BAP1 could inhibit the replication and proliferation of influenza virus in lung,induce the secretion of type I interferons and inflammatory cytokines,and enhance the expression of interferon-stimulated genes.In addition,BAP1 could alleviate the inflammatory cell infiltration of lung tissues and the body weight loss of mice caused by influenza virus infection,prolong the survival time and increase the survival rate of mice infected with lethal dose.Down-regulation of BAP1 significantly inhibited the virus infection-induced phosphorylation of IRF3 while it did not show the significant effects on other signaling pathway molecules in primary macrophages.The luciferase reporter gene experiments showed that BAP1 could effectively enhance the activation of IFN? gene expression induced by IRF3 and its upstream signaling pathway molecules.With the construction of truncations,we found that the activation effect of BAP1 on IFN? gene expression was dependent on its ubiquitination enzyme activity.Furthermore,the experiment indicated that BAP1 could promote IRF3 dimerization and nuclear translocation after viral infection.We discovered that BAP1 could interact with IRF3 among innate immune signaling pathway molecules.We then verified the interaction of endogenous BAP1 with IRF3 in primary macrophages.Next,BAP1 UCH-H region was found to be responsible for the interaction between BAP1 and IRF3 through co-immunoprecipitation experiments.In the following step,we demonstrated that the decreased expression of BAP1 promoted the proteasome degradation of IRF3,and verified that BAP1 could reduce the K48-linked ubiquitination level of IRF3.To further clarify the mechanism,we found the interaction between BAP1 and IRF3 were detected both in cytosol and nucleus,but the binding was stronger in the nucleus with virus infection.It was worth noting that the K48-linked polyubiquitination of IRF3 induced by viral infection mainly occurred in the nuclear component,and BAP1 also mainly inhibited the degradation of IRF3 in the nucleus.Further experiments proved that the BAP1 UCH-H truncation,which could interact with IRF3 and have ubiquitinase activity,could not effectively inhibit the K48-linked polyubiquitination and proteasome degradation of IRF3 in the nuclear due to the disability of nuclear translocation.Since no explicit site has been reported to be responsible for K48-linked polyubiquitination of IRF3,14 potential lysine residues,which are conserved between human and mice,were selected for the construction of Lys-Arg single site mutants.The results suggested that Lys77 and Lys87 were key sites for K48-linked polyubiquitination of IRF3,and the mutation significantly inhibited the degradation of IRF3.The inhibition of IRF3 degradation and the enhancement of IFN? reporter gene activation by IRF3 K77/87 R double-site mutant were more significant than K77 R and K87 R single-site mutants.However,there was no significant difference on IRF3 nuclear translocation induced by viral infection among K77/87 R,K77R and K87 R mutants.Furthermore,it was found that overexpression of BAP1 could downregulate K48-linked polyubiquitination of wild type IRF3 and IRF3 K87 R mutant while there was no significant effect on K77/87 R and K77 R mutants.These results suggested BAP1 specifically removed K48-linked polyubiquitin chains on IRF3 Lys77.In order to further identify the E3 ubiquitin ligase responsible for K48-linked ubiquitination on IRF3 Lys77,we screened reported IRF3 K48-linked ubiquitin ligases,and found that UBE3 C could significantly enhance K48-linked ubiquitination on Lys77,and TRIM26 could enhance K48-linked ubiquitination on Lys87.Further studies discovered that UBE3 C could reverse the inhibitory effect of BAP1 on K48-linked ubiquitination of IRF3.In the early stage of viral infection,BAP1 increased significantly and gradually returned to normal level in the later stage,while the increase in expression level of UBE3 C was later than BAP1 and eventually returned to normal level.Our study suggested that during the phase of acute viral infection,the increased expression of BAP1 inhibited the K48-linked ubiquitination mediated proteasome degradation of IRF3 in nuclear.In the late phase of infection,UBE3 C expression gradually increased and reverse the proinflammatory effect of BAP1,which restrained IRF3 activation to avoid excessive response.In summary,we found that the tumor suppressor gene BAP1 can enhance the antiviral innate immune response by strengthening IRF3 signaling through removing K48-linked polyubiquitin chains on Lys77 of IRF3 and preventing IRF3 from proteasome degradation.Our results also revealed that E3 ubiquitin ligase UBE3 C,together with BAP1,can co-regulate the function of IRF3.The findings further extended the regulatory network of IRF3 expression and functions,providing new insight into the biological function of tumor suppressor BAP1,and outlining a potential target for intervening cancers and viral infections.