| The activation of MAVS,a key adaptor molecule in RIG-I-like receptor（RLR）signaling,is indispensable for antiviral immunity.Yet,the molecular mechanisms modulating MAVS activation are not completely understood.Ubiquitination has a central function in regulating the activity of MAVS.Here,we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS,promotes K63-linked polyubiquitination and subsequent aggregation of MAVS.Besides,USP18 upregulates the expression and production of type I interferon following the infection of Sendai virus（SeV）or encephalomyocarditis virus（EMCV）.Mice with a deficiency of USP18 are more susceptible to RNA virus infection.Mechanistically,USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria.Our results indicate that USP18 functions as a modulator of MAVS-mediated antiviral signaling,suggesting the complexity of post-translational regulation of MAVS.ObjectivesA series of studies have emphasized the importance of posttranslational modification（PTM）in modulating the activity of MAVS.Especially,the studies from our lab and other labs have emphasized that ubiquitination is indispensable for regulating the activation and stability of MAVS.There are abundant numbers of E3 ubiquitin ligases that are responsible for K63-,K27-,and K48-linked polyubiquitination of MAVS,such as TRIM31,TRIM21,and SMURF1.Ubiquitination is a reversible process maintained by both E3 ligases and deubiquitinases（DUBs）.Therefore,DUBs also play significant roles in modulating the turnover and activation of MAVS.However,how these E3 ubiquitin ligases and DUBs cooperate to modulate the ubiquitination level of MAVS in a timely and spatial manner warrants further investigation.Methods and Results1.USP18 enhances MAVS polyubiquitinationWe utilized the Psort Wolf software（https://wolfpsort.hgc.jp/）to predict the possibility of DUBs in mitochondria.Through inputting the protein sequence of approximately 100 DUBs,we found that 13 DUBs are likely distributed in mitochondria over the threshold of 20%.To search for the DUBs involved in the modulation of MAVS ubiquitination,we co-transfected the plasmids encoding HAubiquitin（Ub）,MAVS,and 13 DUBs into HEK293T cells.The polyubiquitination level of MAVS was examined by probing the immunoprecipitates with the HA antibody.Interestingly,we found USP18 significantly enhanced,rather than reduced,the polyubiquitination level of MAVS compared to other DUBs.We examined USP18 expression during RNA virus infection and found that SeV infection greatly increased the human USP18 mRNA and USP18 protein in human THP-1 cells.Similarly,we observed that the level of mRNA and protein of USP 18 was increased in primary peritoneal macrophages（PMs）following SeV infection.To investigate whether USP 18 is associated with mitochondria,we isolated the mitochondria from THP-1 cells according to the protocol from a previous study and found that USP 18 was indeed enriched in the mitochondrial fraction upon SeV infection,which is consistent with the prediction results from Psort Wolf software.More importantly,we observed that the protein level of USP 18 was enhanced in mitochondria rather than ER following RNA virus infection.2.USP18 regulates IFN-β signaling upon RNA virus infectionTo explore the potential role of USP18 in antiviral signaling,we first designed a small interfering RNA（siRNA）that targeted human USP18 and transfected it into human THP-1 cells.We found that expression of endogenous mRNA and protein level of USP18 was much lower in cells transfected with the USP18-specific siRNA than in those transfected with control（non-targeting）siRNA.siRNA knockdown of USP18 expression significantly decreased the expression of IFNB mRNA and downstream CCl5 mRNA in THP-1 cells after SeV infection.Akin to the data obtained with THP-1 cells,we observed that the siRNA knockdown of Usp18 expression in primary mouse macrophages decreased SeV-induced expression of Ifnb and production of IFN-β.Furthermore,the siRNA-mediated knockdown of mouse Usp18 expression in mouse macrophages also decreased EMCV-induced expression of Ifnb and production of IFNβ.Next,we prepared primary peritoneal lacrophages from Usp18+/+ and Usp18-/mice.Consistent with the observation from the siRNA knockdown of USP18,infection of Usp18-/-macrophages with SeV led to a decrease in fold changes of Ifnb mRNA as well as the production of IFN-β compared with Usp18+/+ macrophages.Congruently,the fold changes of Ccl5 mRNA from Usp18-/-peritoneal macrophages were also declined compared with Usp18+/+counterparts.Infection of Usp18-/-peritoneal macrophages with EMCV also led to a decrease in fold change of Ifnb and its downstream Ccl5 gene mRNA levels as well as the production of IFN-β compared with the WT counterparts.To further validate the effect of USP18 in other cell types,we isolated the primary MEFs from Usp18+/+ and Usp18-/-mice and infected them with either SeV or EMCV.Akin to the phenomenon we observed in primary peritoneal macrophages,Usp18-/-MEFs exhibited significantly impaired expression of Ifnb and its downstream genes as well as the production of IFN-β compared with Usp18+/+MEFs.The positive regulation of USP18 in the RLR pathway was independent of RNA viral infection,since the deletion of USP18 in MEFs significantly impaired the expression of Ifnb after transfection of viral RNA analog Poly（I:C）LMW and HMW,which are the ligands for RIG-I and MDA5 respectively.We pretreated the MEF cells with an anti-IFNAR antibody,which can efficiently block the induction of IFN downstream genes,as indicated by the much weaker induction of Usp18 expression upon SeV infection.We observed that pretreatment with IFNAR antibody still resulted in a significant difference between Usp18+/+ and Usp18/-cells followed by SeV infection.However,we observed that the difference was smaller than that with isotype antibody treatment.Furthermore,we found that pretreatment of MEF cells with mouse recombinant IFN-β led to an enhanced difference between Usp18+/+ and Usp18-/-cells following SeV infection.We analyzed changes in the phosphorylation of TBK1 and IRF3,which are the downstream events of MAVS activation and hall marker of interferon pathway activation.Phosphorylated TBK1（pTBK1）and IRF3（pIRF3）were efficiently induced after SeV infection in Usp18+/+ MEFs,while these signals were significantly lower in Usp18-/-MEFs.Since phosphorylated IRF3 led to the dimerization of IRF3,we then examined changes in the dimerization of IRF3.We observed that Usp18-/-MEFs exhibited less dimerized IRF3 compared with Usp18+/+ MEFs.3.USP18 regulates RNA virus infectionTo investigate the role of USP18 in the regulation of the viral infection,we measure the SeV viral protein using the SeV antibody in MEF cells after infection with SeV.The band with the strongest intensity corresponds to SeV P protein according to the molecular weight.Consistent with the function of USP18 in the regulation of IFNβ signaling,we observed an enhanced viral protein level of SeV in Uspl8-/-MEFs compared to that in Usp18+/+ MEFs.Furthermore,overexpression of USP18 in HEK293T cells resulted in an increased level of pIRF3 and correspondingly reduced level of SeV in a dosage-dependent manner.This phenomenon is not due to the transfection of plasmids since the overexpression of GFP in HEK293T cells could not affect the levels of pIRF3 and SeV.To examine the physiological relevance of USP18 during RNA virus infection in vivo,we challenged Usp18+/+ and Usp18-/-mice with VSV-GFP.To exclude the possibility that USP18 may affect the development of the immune system,we isolated the thymus,spleen,and peripheral lymph glands from Usp18+/+ and Usp18-/-mice and observed that there was no substantial distinction between them.We found,by ELISA analysis,that the production of IFN-β in the serum of Usp18-/-mice was significantly lower than that of Usp18+/+ mice after infection with VSV.Moreover,we observed a more severe paralysis symptom in Usp18-/-mice after infection,with a corresponding higher VSV titers in the brain.We also observed that VSV titers and VSV-GFP protein were significantly greater in the spleen,liver,and lung of Usp18-/-mice than in those from Usp18+/+mice.Furthermore,Usp18-/-mice were less resistant to infection with VSV compared with Usp18+/+ mice through intraperitoneal injection,exhibiting significantly reduced survival time.Furthermore,hematoxylin-and-eosin staining showed greater infiltration of immune cells and injury in the lungs of Usp18-/-mice,relative to that in the lungs of Usp18+/+ mice,after infection with VSV.4.USP18 interacts with MAVS in the RLR signaling pathwayTo investigate whether USP18 specifically interacts with MAVS,we utilized the Co-IP assay to examine the interaction between the signaling molecules in the RLR signaling pathway and USP18.The results showed that USP18 mainly interacted with MAVS,while there was no or weak association with upstream molecules RIG-I or MDA5 and downstream molecules TBK1 or IRF3 in the RLRs signaling pathway.We also observed that the endogenous interaction between USP18 and MAVS was enhanced following the SeV infection in THP-1 cells.The elevated endogenous interaction between MAVS and USP18 was also confirmed in the RAW264.7 cells upon SeV infection.we found that USP18 interacted with the fragment of 173 aa to 513 aa in MAVS,located between the proline-rich and TM domain.Immunofluorescence assay showed that Myc-USP18 exhibited colocalization with Flag-MAVS.The spatial colocalization and interaction between USP18 and MAVS were further confirmed by in situ proximity ligation assay（PLA）,in which the number of red spots displaying the interaction between USP18 and MAVS.There were no red spots when Myc-USP18 was co-transfected with the control Flag vector,while the transfection of both Myc-USP18 and Flag-MAVS resulted in significant numbers of red spots.The number of spots representing the endogenous USP18-MAVS complex increased significantly following SeV infection.We further isolated the mitochondrial fraction and observed an enhanced interaction between MAVS and USP18 in mitochondria following SeV infection.5.USP18 enhances the K63-linked polyubiquitination and aggregation of MAVSNext,we investigated whether USP18 specifically modulates the ubiquitination level of MAVS,we transfected plasmids encoding USP18,ubiquitin together with signaling molecules in the RLRs pathway into HEK293T cells.We observed that USP18 only enhanced the ubiquitination level of MAVS rather than RIG-I,MDA5,and TBK1.Consistent with the results from overexpression of USP18,siRNA knockdown of USP18 in RAW264.7 cells resulted in a decrease of K63-linked ubiquitinated MAVS.Through transfecting K48-or K63-specific linkage Ub mutant,we observed that USP18 enhanced the K63-linked,instead of K48-linked,polyubiquitination of MAVS.Overexpression of USP18 in HEK293T cells did not significantly affect the protein level of endogenous MAVS.Besides,knockout of USP18 in mouse embryonic fibroblast（MEF）cells did not affect the MAVS protein level with the infection of SeV.Importantly,the turnover rate of MAVS was comparable between Usp18+/+ and Usp18/-MEFs under the treatment of Cycloheximide（CHX）.Since IP assay likely precipitates the ubiquitination proteins that interact with MAVS,we also utilized the TUBE assay with GST-K63-TUBE,which enrich the K63linked ubiquitinated proteins.We observed that MAVS of Usp18-/-MEF displayed significantly less K63-linked polyubiquitination following viral infection compared with that of Usp18+/+ MEFs.Semi-Denaturating Detergent Agarose Gel Electrophoresis（SDD-AGE）analysis showed that overexpression of USP18 resulted in more formation of MAVS aggregates compared with the control Myc vector.Furthermore,we observed that the transfection of USP18 together with MAVS in HeLa cells led to more MAVS aggregate,exhibiting decreased mitochondrial skeleton length and cytosol area.Consistent with the observation from overexpression of USP18,Usp18-/-MEF displayed decreased aggregation of MAVS demonstrated by both SDD-AGE and immunofluorescence assays compared with Usp18+/+ MEFs upon SeV infection.6.USP18 modulates the ubiquitination of MAVS independent of its enzymatic activityWe mutated the cysteine residue 64 of USP18 to serine（USP18C64S）.We observed that the interaction between MAVS and USP18 was not affected by this mutation.Furthermore,we observed that USP18C64S could enhance the K63-linked polyubiquitination of MAVS like wild-type USP18.Consistently,USP18C64S resulted in the aggregation of MAVS to the extent comparable to wild-type USP18 through both SDD-AGE and immunofluorescence assays.We also found that transfection of both USP18 and USP18C64S into HEK293T cells led to an increase of phosphorylated IRF3 and subsequently a decrease of SeV protein in a dosage-dependent manner.7.USP18 promotes MAVS K63-linked polyubiquitination dependent on E3 ligase TRIM31We observed that knockdown of TRIM31 in HEK293T cells led to the abrogation of the enhanced K63-linked ubiquitination of MAVS by USP18.Furthermore,overexpression of USP18 in Trim31+/+ MEFs,rather than Trim31-/-MEFs,led to an enhanced K63-linked polyubiquitination of MAVS.To further delineate the relationship between USP18 and TRIM31 in regulating the ubiquitination of MAVS,we knocked down USP18 in the presence or absence of TRIM31.We observed that ablation of USP18 in the presence of TRIM31 could decrease the K63-linked polyubiquitination level of MAVS,while knockdown of USP18 in absence of TRIM31 could not further downregulate the ubiquitination level of MAVS.We found that K63-linked polyubiquitination of MAVS K1OR,K311R,and K461R cannot be enhanced by USP18 compared with WT MAVS.We found that knockdown of USP18 could lead to a decrease of IFNB,CCL5,and CXCL10 in MAVS KO HeLa reconstituted with WT MAVS instead of MAVS 3KR following SeV infection.8.USP18 promotes the interaction between MAVS and TRIM31 in mitochondriaThe overexpression of USP18 in HEK293T cells did not affect the protein level of TRIM31.We observed that USP 18 did interact with TRIM31 through the Co-IP assay.Immunofluorescence assay showed that USP18 can colocalize with TRIM31.More importantly,we observed that the interaction between endogenous USP18 and TRIM31 was increased following SeV infection.We observed that USP18,TRIM31,and MAVS form a complex through Co-IP assay.We observed that TRIM31 was efficiently enriched in mitochondria in Usp18+/+ MEFs following SeV infection.As a control,the MAVS level in mitochondrial fraction was not affected in the presence or absence of USP18.We observed that the interaction between MAVS and TRIM31 following viral infection was significantly impaired in Usp18-/-MEFs compared with Usp18+/+ MEFs.Furthermore,overexpression of USP18 could enhance the interaction between MAVS and TRIM31 in a dosage-dependent manner.Interestingly,we also observed that the addition of USP18 in the in vitro ubiquitination assay did not enhance the ubiquitination of MAVS catalyzed by TRIM31 different from the ubiquitination assay within cells further indicating that USP18 very likely bridges the link between TRIM31 and MAVS in vivo.ConclusionIn this study,we utilize Psort Wolf software to predict the DUBs likely located in mitochondria.We find 13 DUBs are likely distributed in mitochondria.Interestingly,we observe that USP18 can significantly promote the polyubiquitination of MAVS.The interaction between USP18 and MAVS is enhanced following viral infection,suggesting the possible regulation of MAVS activity by USP18.Furthermore,USP18 facilitates the K63-linked polyubiquitination and subsequent aggregation of MAVS.Consequently,the deletion of USP18 in macrophages or fibroblasts leads to impaired type Ⅰ interferon response following viral infection.More importantly,mice with a deficiency of USP18 are more susceptible to viral infection.Mechanistically,the effect of USP18 on MAVS is independent of its enzymatic activity but dependent on TRIM31.Upon viral infection,USP18 is critical for the re-localization of TRIM31 from the cytoplasm to mitochondria and the interaction between TRIM31 and MAVS.Innovation1.Previously,USP18 is identified as a negative regulator of type Ⅰ interferon signaling via inhibiting the interaction between JAK and IFNAR.Consequently,USP18 deficiency enhances viral resistance after type Ⅰ IFN stimulation.Our study indicates USP18 plays different roles in different steps of type Ⅰ interferon signaling.2.It is an intriguing observation that USP18,as a DUB,enhanced the polyubiquitination of MAVS.3.Following viral infection,USP18 is enriched in mitochondria and promotes the mitochondrial translocation of TRIM31 from the cytoplasm and interaction between TRIM31 and MAVS,consequently enhancing TRIM31-mediated K63-linked polyubiquitination and subsequent aggregation of MAVS.Our study suggests a complicated regulatory mechanism of MAVS aggregation,emphasizing its supreme importance in the innate antiviral response. |
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