Function And Molecular Mechanism Of USP27X In The Innate Antiviral Immunity Against DNA Virus

Pattern recognition receptors(PRR)can stimulate innate immunity by recognizing pathogen associated molecular patterns(PAMP).In this process,the PRRs are activated by its conserved regions in combination with pathogen-associated molecular patterns,which in turn induces the expression of antiviral proteins and pro-inflammatory factors to achieve the body’s innate immune clearance of pathogenic microorganism infection.Cyclic GMP-AMP synthase(cGAS),a cytosolic DNA sensor,catalyzes the formation of the second messenger 2’3’-cGAMP which can bind to STING and triggers the type I IFN signaling.Activation of cGAS can be modulated by several protein posttranslational modifications,including ubiquitination.However,the cGAS activation regulated by protein deubiquitination remains poorly understood.In this study,we identified that deubiquitinase USP27X could interact with cGAS and cleave K48-linked polyubiquitination chains from cGAS,leading to cGAS stabilization.Consistently,knockout of Usp27x in mice macrophages resulted in an accelerated turnover of cGAS,decreased cGAMP production,phosphorylation of TBK1 and IRF3,and IFN-P production.Furthermore,Usp27x knockout mice macrophages showed impaired innate antiviral responses against HSV type 1 infection.Our data suggest that USP27X is a novel regulator of the cGAS-STING cytosolic DNA sensing pathway.Methods amd Results:To investigate the regulation of cGAS deubiquitination and test whether the DUB affects cGAS-mediated anti-DNA virus signaling pathways and further to clarify the specific molecular regulatory mechanism.1.USP27X regulates cGAS deubiquitination through its deubiquitinase activitywe overexpressed DUB plasmids into HEK293T cells together with V5-cGAS and HA-Ub plasmids,then the ubiquitination of cGAS was analyzed through IP and Western blotting.The result shows that USP27X can significantly regulate cGAS ubiquitination.Exogenous ubiquitination experiments were performed by transfecting Flag-tagged cGAS and HA-tagged Ub,together with Myc-tagged wild-type USP27X or its active mutant USP27X C87A,and IP was performed with Anti-Flag beads.cGAS ubiquitination level was detected by western blot.The results suggest that USP27X can indeed deubiquitinate cGAS which rely on its deubiquitinase activity.To exclude any artifacts caused by overexpression and to determine whether this regulation occurs in physiological conditions,we performed endogenous deubiquitination experiments by using interfering RNA to knock down Usp27x in peritoneal macrophage.We used HSV-1 to stimulate cells for indicated points,the lysate was immunoprecipitated with anti-cGAS antibody,cGAS ubiquitination level was detected by western blot.The results suggest that knocking down Usp27x significantly increased cGAS endogenous ubiquitination level.In addition,we repeated this experiment by Usp27x KO RAW264.7 and reached the same conclusion.In order to prove that the deubiquitination of cGAS is directly regulated by USP27X,we performed in vitro deubiquitination experiments.The Usp27x WT or Usp27x C87A protein translated in vitro was added to the enriched ubiquitinated cGAS,the cGAS ubiquitination level was detected after 1 hour incubation at 37℃.The results suggest that USP27X can directly deubiquitinate cGAS which is related to its deubiquitinating enzyme activity.2.USP27X regulates cGAS by interacting with cGAS2.1 In order to investigate whether USP27X regulates cGAS deubiquitination through interacting with it,we first transfected 293T cells with Flag-cGAS and Myc-tagged USP27X.Anti-Flag beads was used for IP,and Western blot was used to detect whether USP27X could interact with cGAS.To further clarify the interaction between the two,we performed endogenous binding experiments.HSV-1 infected macrophages at different time points.IgG or Anti-cGAS antibody were selected for IP,and Western blot was used to detect the binding.The result suggests that endogenous USP27X can interact with cGAS,and the degree of binding is significantly enhanced with the increase of stimulation time.2.2 After clarifying the interaction between the two,we further explored whether the binding between the two is direct.We perform co-immunoprecipitation experiments with Myc-USP27X and Flag-cGAS proteins.The results suggest the combination of USP27X and cGAS is direct.3.USP27X stabilizes cGAS3.1 In order to explore which ubiquitination form of cGAS is mainly regulated by USP27X,we performed an exogenous CO-IP experiment.First,we overexpressed Flag-tagged cGAS and WT HA-tagged Ub or its mutants with different lysine residue sites(K6,K11,K27,K29,K33,K48,K63)in HEK293T cells together with USP27X.and IP was performed by anti-Flag beads and cGAS ubiquitination was detected by western blot.The results suggest that USP27X mainly catalyzes K48 linked polyubiquitination of cGAS.3.2 K48 linked ubiquitination mainly mediates substrate degradation.We analyzed cGAS protein levels.Gradient overexpression of Myc-USP27X or Myc-USP27X C87A were overexpressed in HEK293T cells and cGAS protein levels were detected by western blot.With gradient overexpression of USP27X,cGAS protein levels significantly increase.We also examined the regulation of cGAS by endogenous USP27X through interfering RNA and knockout cell lines.The results suggest that interference with Usp27x can significantly reduce cGAS protein levels.Consistent with this,cGAS protein levels of Usp27x knockout macrophages were significantly lower than those of the control group under HSV-1 stimulation.The above shows that USP27X can indeed affect cGAS protein levels.3.3 To exclude the effect of synthesis on cGAS protein levels,we performed a half-life test.After stimulating WT and Usp27x KO RAW264.7 cells for 4 hours with DNA,cells was treated for 0,4,8,12 hours with CHX,and cGAS protein levels were detected by western blot.The results showed that the degradation rate is significantly accelerated when knocking out Usp27x.3.4 We designed four groups:WT;Usp27x KO RAW264.7;Usp27x KO RAW264.7+ MG 132(proteasome degradation inhibitor);Usp27x KO RAW264.7+CQ(autophagolysosomal degradation inhibitor).The results suggest that both proteasome and lysosomal degradation pathways exist in cGAS in the resting state,but autophagy lysosomal degradation is mainly performed in the case of viral infection,and USP27X mainly regulates cGAS autophagolysosomal degradation pathway upon DNA virus infection.4.USP27X regulates cGAS-mediated DNA signaling pathway4.1 Activation of the entire DNA signaling pathway depends on phosphorylation of TBK1 and dimerization of IRF3.Therefore,we tested the phosphorylation and dimerization of downstream TBK1 and IRF3 to reflect the regulation of USP27X on the DNA signal pathway.First,we interfered Usp27x in primary peritoneal macrophages,followed by DNA pathway stimulants treatment:HSV-1,ISD for 0,4,and 8 hours.The results suggest that knocking down USP27X,phosphorylation of TBK1,IRF3 induced by HSV-1 and ISD is significantly reduced,and IRF3 dimerization is significantly reduced.To prove that USP27X regulates the DNA pathway by regulating cGAS rather than other sensors,we used cGAMP stimulation as a control.In contrast,cGAMP-induced phosphorylation of IRF3 and TBK1 was barely affected in Usp27x-KO RAW264.7 cells and WT cells.We also tested the RLR signaling pathway.The results suggest that interference with Usp27x can significantly increase the phosphorylation of TBK1 and IRF3 induced by SeV or poly(I:C).To further clarify the role of USP27X in regulating the DNA pathway,we performed experiments using a knockout cell line and got the same conclusion.4.2 USP27X deficiency inhibits the production of IFN-β caused by DNA viruses.To further explore the functions and physiological significance of USP27X in the antiviral pathway.We analyzed the production of IFN-p.Knockout of Usp27x inRAW264.7 macrophages resulted in decreased expression of Ifnbl mRNA expression on ISD transfection and HSV-1 infection.In contrast,cGAMP stimulation-induced Ifnbl mRNA expression was comparable in si Usp27x and si scramble cells.Interferon needs to be secreted for its antiviral function.We tested the IFN-β secretion level by ELISA.The results suggest that knockdown of USP27X,HSV-1,and ISD induced a significant decrease in IFN-β secretion,but no significant change in cGAMP-induced IFN-β secretion.To further clarify the regulatory effect of USP27X on the DNA pathway,we performed experiments using a knockout cell line.Knockout of Usp27x in RAW264.7 macrophages resulted in decreased expression of Ifnbl mRNA expression on ISD transfection and HSV-1 infection.In contrast,cGAMP transfection-induced Ifnb1 mRNA expression was comparable in Usp27x-KO and WT cells.In addition,we also performed rescue experiments to determine the regulatory effect of Usp27x on the DNA pathway.We designed four groups of experiments:WT,Usp27x KO RAW264.7,Usp27x KO RAW264.7+lenti-USP27X WT;Usp27x KO RAW264.7+lenti-USP27X C87A.The results suggest that the over expressed wild-type Usp27x lentivirus can significantly complement the phosphorylation levels of TBK1 and IRF3,while the over-expressing mutant Usp27x C87A lentivirus has no significant complementing effect.Overexpression of WT Usp27x,but not the enzymatic mutant C87A can rescue HSV-1 infection-induced expression of Ifnbl mRNA and IFN-β secretion in Usp27x-KO RAW264.7 cells.Collectively,these data indicated that USP27X is essential for the cytosolic DNA-induced innate signaling and IFN-b production.5.Effect of USP27X on cGAS synthetase activity.To explore the function of USP27X-mediated deubiquitination and stability of cGAS,we measured cGAMP production in Usp27x-KO RAW264.7 cells after transfection with HSV-60.cGAMP levels in the stimulated cells were indirectly measured through IRF3 phosphorylation and dimerization.As expected,culture supernatant from WT macrophages stimulated with HSV-60 induced IRF3 phosphorylation and dimerization,whereas the culture supernatant from Usp27x-KO RAW264.7 induced less IRF3 phosphorylation and dimerization,indicating Usp27x-KO RAW264.7 cells produced less cGAMP on HSV-60 transfection compared with WT macrophages.We also measured the cGAMP production in Usp27x-KO cells after HSV-60 transfection using 2939-cGAMP ELISA Kit.Similar to the data of cGAMP-induced IRF3 activation,cGAMP production was decreased in Usp27x-KO cells comparedwith those in WT macrophages.Importantly,restoration of the WT Usp27x expression,but not the mutant C87A expression rescued HSV-60-induced cGAMP production in Usp27x-KO cells.6.USP27X Antiviral Physiological SignificanceFinally,we assessed if USP27X played a role in cellular antiviral response.WT and Usp27x-KO RAW264.7 cells were infected with HSV-1,and then the virus titer in the culture medium and genomic DNA copy of HSV-1 in the infected cells were measured by plaque assay and quantitative PCR,respectively.As a control,VSV was also used to infect the WT and Usp27x-KO cells.Plaque assay of RAW264.7 cells infected with HSV-1 showed that knockout of Usp27x greatly increased viral replication compared with that in WT infected cells.Consistently,the HSV-1 genomic DNA copy number was also significantly increased in the Usp27x-KO cells compared with WT cells.Importantly,restoration of WT Usp27x expression,but not the mutant C87A expression in Usp27x-KO cells,repressed HSV-1 replication.Knockdown of Usp27x expression in primary peritoneal macrophages through Usp27x siRNA also increased HSV-1 replication.In contrast,VSV replication and VSV virus titer was decreased significantly.The results suggest that USP27X plays a positive regulatory role in anti-DNA viruses,but mainly plays a negative regulatory role in anti-RNA virus pathways.Conclusions and innovative topics1.We screened five DUB families and detected that USP27X can directly interact with cGAS and directly deubiquitinate cGAS K48 linked ubiquitin chains.At present,there are few studies on cGAS deubiquitination.Our research fills this gap and provide a new direction for cGAS function exploration.2.We specifically explored the degradation of cGAS,that is,cGAS mainly undergoes proteasome degradation and autophagolysosomal degradation in the resting state,but in the case of viral infection,cGAS mainly performs autophagolysin degradation in vivo.Moreover,the deubiquitination regulation of cGAS by USP27X mainly mediates the autophagolysosomal degradation pathway in its viral infection state.3.It has been shown that USP27X plays an important role in the antiviral pathway.USP27X is related to life activities such as apoptosis,but its function in innate immune immunity has not been explored.Our study explored in detail that how USP27X played a positive role in regulating antiviral pathways and enhanced the ability of cells to resist DNA viruses through the use of knockout cell lines and the use of rescue assay by over-expressing lentivirus.4.Our research provides new targets for clinical antiviral therapy.Viral infection is still a major problem facing the world,and its heterogeneity has increased the difficulty of clinical treatment.The carrer rate of HSV-1 herpes virus is also very high.It is extremely harmful to the human body which can induce cold sores,keratitis and even encephalitis,which seriously endangers human physical and mental health.We have found that USP27X can exert antiviral functions by regulating cGAS deubiquitination to mediate IFN-β production.This study provides new ideas and new targets for clinical antiviral infection treatment.