The Molecular Mechanisms Of Ubiquitin-Specific Protease 30 Degrades TbBK1 To Facilitate Virus Replication

The innate immune system serves as the first line of defense against viral invasion by detecting the pathogen-associated molecular patterns（PAMPs）.Viral RNA and DNA can be primarily recognized by several classes of pattern-recognition receptors（PRRs）,these PRRs trigger the activation of NF-κB,IRF3/7,and inflammasome signaling pathways through distinct adaptor proteins,leading to the production of typeⅠIFNs and pro-inflammatory cytokines.Secreted typeⅠIFNs bind to the IFNα/βreceptor（IFNAR）to activate the JAK/STAT signaling pathway,triggering the expression of antiviral genes,and finally inhibits viral replication,triggering the antiviral immune response.TBK1,which belongs to the I-kappa-B kinase（IKK）family,is a critical kinase for the activation of IRF3 and IFN-βproduction in type I IFN signaling,and its activity is tightly regulated by multiple post-translational modifications（PTMs）,such as phosphorylation,sumolytation and ubiquitination.USP30,a member of the USP family,is a transmembrane DUB located in the mitochondrial outer membrane that inhibits mitophagy mediated by ubiquitin-ligase PARK2 and protein kinase PINK1,and also prevents pexophagy by inhibiting the E3 ubiquitin ligase activity of PEX2.However,the role of USP30 in innate immunity has not been reported.In this study,the innate immune signaling pathways regulated by USP30 were taken as an entry point to explore the molecular mechanism of the degradation of TBK1by USP30 and the promotion of viral replication.The specific contents are as follows:（1）By using CRISPR/Cas9 gene editing technology,we achieved efficient editing of USP30 gene and successfully constructed a USP30 knockout HEK-293T cell line.（2）The dual luciferase reporting system is used to demonstrate that USP30significantly inhibited IFN-βand ISRE promoter activity induced by Poly（d A:d T）,Poly（I:C）,Se V and VSV.Real-time quantitative PCR experiments confirmed that USP30 could significantly inhibit the expression of IFN-β,ISG15 and ISG54 m RNA induced by Poly（d A:d T）,Poly（I:C）,Se V and VSV.USP30 knockdown or knockout significantly increased the activities of IFN-βand ISRE promoter,and the expression of IFN-β,ISG15 and ISG54 m RNA were significantly higher than those of wild-type cells.Overexpression of USP30 can significantly reduced the IFN-βsecretion induced by Se V infection.USP30 knockout significantly increased the IFN-βsecretion induced by Se V infection.We also noticed that macrophages from USP30^-/-mice have higher protein secretion of IFN-β.Meanwhile,we found that USP30 markedly inhibited the phosphorylation of TBK1 and IRF3 upon treatments of Se V.USP30 knockout markedly enhanced the phosphorylation of TBK1 and IRF3 induced by Se V infection.And HSV-1-induced phosphorylation of IRF3 and TBK1 was markedly enhanced in USP30^-/-MEFs compared with the wild-type counterparts.These results indicated that USP30 can inhibit type I interferon signaling pathway.（3）The dual luciferase reporting system is used to demonstrate that USP30significantly inhibited activation of luciferase reporter by c GAS+STING,MDA5,RIG-I,MAVS and TBK1.IRF3-5D induced luciferase reporter activation remains unchanged by USP30 expression.Co-immunoprecipitation and immunoblot analysis revealed that USP30 specifically interacted with TBK1.In addition,confocal microscopic analysis showed that USP30 co-localized with TBK1 in the cytosol.And Co-immunoprecipitation and immunoblot analysis revealed that the C-terminal USP domain of USP30 interacted with TBK1.（4）Further study indicated that USP30 inhibit the expression of TBK1 in a dose-dependent manner.And USP30-induced TBK1 degradation is dependent on a lysosome pathway.The USP30 C77S mutant failed to degrade TBK1,indicating that the deubiquitinating activity of USP30 is required to degrade TBK1（5）To explore the molecular mechanism that USP30 degrade TBK1,we found that USP30 interacted with BAG2 in overexpression and coimmunoprecipitation assays.And USP30 significantly reduced TBK1 abundance in the presence of BAG2,while knockdown of BAG2 markedly enhanced the abundance of TBK1 upon treatments of Se V.Consistent with this result,BAG2 enhanced the USP30-mediated degradation of TBK1 and inhibited IFN-β-luc and ISRE-luc activity.（6）The dual luciferase reporting system and real-time quantitative PCR experiments is used to demonstrate that BAG2 significantly reduced TBK1 abundance through the autophagolysosome pathway.Co-immunoprecipitation and immunoblot analysis revealed that BAG2 specifically interacted with TBK1.In addition,confocal microscopic analysis showed that BAG2 co-localized with TBK1 in the cytosol.BAG2reduced the expression of IFN-β,ISG15,and ISG54 mRNA induced by Se V,while in USP30^-/-HEK293T cells restored the m RNA level.In conclusion,we first successfully constructed a USP30 knockout HEK-293T cell line,clarified the role of the host protein USP30 targeted degradation of TBK1 to inhibit the activation of type I interferon signaling pathway and promote viral replication,and explored the mechanism of USP30 to decrease the expression of TBK1.The results of this study will deepen the understanding of the new function of USP30 and the negative feedback mechanism of the host innate immune response against the virus,and provide a theoretical basis for the research development of antiviral drugs.