Usp18 Recruits USP20 To Promote Innate Antiviral Response Through Deubiquitinating MITA

The innate immune system is the first line of host defense against infection of microbial pathogens.Host cells express various of germline-encoded pattern-recognition receptors?PRRs?to detect structurally conserved molecules pathogen-associated molecular pattarns?PAMPs?.After binding to PAMPs,PRRs recruit and activate downstream adaptor proteins and kinases to trigger a series of signaling cascades that lead to the expression of type I interferons?IFNs?and cytokines.Type I interferons then induce the expression of antiviral genes to defense viral infection and clear the infected cells.PRRs can be divided into four groups according to their structure,location and function,including Toll-like receptors?TLRs?,cytosolic RIG-I-like receptors?RLRs?,cytosolic NOD-like receptors?NLRs?and cytosolic DNA sensors.The nucleotidyl transferase cyclic AMP-GMP synthase?cGAS?has been identified as a new cytosolic DNA sensor,which is critical for host defense against DNA viruses.cGAS binds to DNA to form a 2:2 complex that induces a conformational change in the active site,which catalyzes the synthesis of cyclic GMP-AMP?cGAMP?from ATP and GTP.cGAMP functions as a second messenger and binding to the endoplasmic reticulum?ER?and mitochondrial adaptor protein MITA?also called MITA,MPYS and ERIS?that results in the protein transportation and activation of MITA.MITA further recruit downstream adaptor proteins and kinases such as TBK1/IRF3 or TRAF6/IKK complex to activate IRF3 and NF-?B signaling pathway that induce the expression of antiviral genes including type I interferons?IFNs?,TNF,IL6 and CCL5.Virus infection results in the post-translational modifications that is also essential for cellular antiviral responses.MITA is ubiquitinated by some E3 ubiquitin ligase,TRIM32 and TRIM56catalyzes K63-linked ubiquitination of MITA,AMFR-INSIG1 E3 ligase complex mediates K27-linked polyubiquitin of MITA,RNF26 catalyzes K11-linked ubiquitination of MITA,all these E3 ubiquitin promotes MITA induced pathway.In contrast,RNF5 and TRIM29 catalyze K48-linked ubiquitination of MITA,causing the proteasome-dependent degradation of MITA and thus turning down the excessive immune responses.The ubiquitinations of MITA determine the role of MITA in innate immune response.Neverthless,the deubiquitination of MITA is rarely revealed.Deubiquitination is the reverse process of ubiquitination and mediated deubiquitinating enzymes.Ubiquitin-specific protease?USP?18 and USP20 belong to the USP subfamily of DUBs and mediate deubiquitination of targets.USP18 was also characterized as a deISGylation enzyme,enzyme-inactive mutant USP18?C61A?in mice results in hyper-ISGylation.Furthermore,USP18 binds to interferon?receptor?IFNAR?subunit IFNAR2 that blocks JAK1 binding to IFNAR2 and negatively regulates type I interferons induced JAK-STAT signaling pathway.USP20 has been reported involved the deubiquitination of various proteins that regulates IL-1?signaling,DNA damage repair,tumorigenesis,and chemoresistance of cancers.USP18 and USP20 have never reported relate to virus-triggered innate immune response signaling pathway.In this study,we identified USP18 recruits USP20 to mediate the innate antivral signaling by deubiquitinates the adaper protein MITA.USP18 deficiency impaired DNA virus-or cytoplasmic DNA-triggered activation of IRF3 and NF-?B and subsequent induction of type I IFNs and pro-inflammatory cytokines.In addition,USP18^-/-mice were more susceptible to HSV-1 infection.Reconstitution of either USP18 or USP18?C61A?into USP18^-/-MEFs restored DNA virus-or cytosolic DNA-induced activation of innate immunity.The enzymatic activity of USP18 is dispensable for virus-triggered induction of type I IFNs.Further results shows that USP18 interacts with and recruits USP20 to MITA,and USP20 mediates deconjugation of K33/K48-linked polyubiquitin chains from MITA.Knockdown of USP20 resulted in hyperubiquitination and accelerated degradation of MITA as well as increased replication of HSV-1.Our findings thus uncover a previously uncharacterized role of USP18 and USP20 in regulating virus-triggered MITA-mediated type I IFN induction and contribute to the complicated regulatory mechanisms of innate cellular antiviral responses.