Regulatory Mechanism Of Hepatitis B Virus X Protein On CGAS Signaling Pathway

Objective To explore the mechanism that HBx protein inhibits cGAS mediated DNA sensing pathway.Methods 1.HEK293 cells were co-transfected with pIFN?-luc,pRL-PK and pcGAS-Flag,pSTING-HA,pHBX plasmid,respectively.Cell lysates were collected after transfection for 24 hours,and luciferase activity was detected by multi-functional enzyme analyzer.2.HEK293 cells were co-transfected with pcGAS-Flag,pSTING-HA and pHBX plasmids.IFN? mRNA was detected by real-time fluorescence quantitative PCR at various time points.3.The protein and mRNA expression levels of cGAS and STING in HepG2,HepG2.215,L02 and SMMC-7721 cell were detected by Western blot and real-time PCR.4.HEK293 cells were co-transfected with pcGAS-Flag,pSTING-HA and pHBX plasmids,and proteins were extracted and analyzed for expression of phosphorylated and dimeric forms of IRF3 by Western-blot.5.HEK293 cells were co-transfected with pIRF3-Flag and pSTING-HA with pHBX plasmids,and cell lysates were collected 24 hours after transfection.The relative luciferase activity was detected by multi-functional enzyme analyzer.6.SMMC-7721 cells were transfected with pHBX plasmid.The protein was extracted and analyzed by Western-blot to analyze the effect of HBX on cGAS protein expression,the RNA was extracted and the effect of HBX on cGAS mRNA expression was analyzed by real-time PCR.7.SMMC-7721 cells were transfected with pHBX plasmid.The combination of HBX and cGAS was detected by co-immunoprecipitation and the colocalization was observed by fluorescence microscopy.8.SMMC-7721 cells were treated with 3MA and Rapamycin or SMMC-7721 cells were transfected with pHBX plasmid to detect the changes of cGAS and LC3-II protein expression;SMMC-7721 transfected with pGFP-LC3 plasmid were treated with 3MA or Rapamycin,alternatively,pGFP-LC3 and pHBX were co-transfected in SMMC-7721 cells,autophagic particles were observed under a fluorescence microscope,the number of autophagy cells was counted,and the incidence of autophagy was calculated.Results 1.HBX negatively regulates the expression of IFN? induced by cGAS.2.HepG2,HepG2.215,L02 and SMMC-7721 cell express STING,but onlyL02 and SMMC-7721 cells express cGAS.3.HBX could inhibit the signal of cGAS-STING-IRF3-IFN? pathway,and it mainly happened at the upstream of STING.4.HBX has an inhibitory effect on the protein expression and function of cGAS,but has no effect on the mRNA level of cGAS.5.HBX co-localized with cGAS in cells and has ability to combine cGAS.6.HBX promotes the degradation of cGAS by enhancing the autophagy,thereby negatively regulating the cGAS signaling pathway.Conclusion HBX might negatively regulate cGAS-mediated DNA signaling through two mechanisms.On the one hand,HBX combine cGAS and competitively inhibit its binding to DNA.On the other hand,HBX enhances the autophagy of cGAS which promotes the degradation of cGAS.