The Effect Of Double-Stranded DNA On Replication Of Hepatitis B Virus And Cellular Signaling

ObjectiveTo explore the possibility that dsDNA may change the intracellular innate immuneresponses and therefore regulate the replication of unrelated viruses like hepatitis B virus(HBV) and the role of DAI(DLM-1/ZBP1) plays in typeⅠinterferon response to dsDNA.Methods1. A hepatoma cell line HepG2 and its derivate with HBV, HepG2.2.15, were treated withpoly(dA-dT)·poly(dT-dA), a synthetic double-stranded DNA. Cellular total RNAs wereextracted and detected for IFN-β, IFIT1, and TNF-αexpression by reverse transcription realtimePCR. The translocation of IRF3 and NF-κB and the phosphorylation of TBK1 weredetected by western blot.2. HepG2 cells were transfected with HBV replicative plasmid of pHY106+wta, orHepG2215 cells were transfected with small interfering RNA of HBV(HBV siRNA). Afterpoly(dA-dT)·poly(dT-dA) stimulation, the expression of IFIT1 in the both cell lines weredetected by real time RT-PCR.3. HepG2215 cells were transfected with poly(dA-dT)·poly(dT-dA). HBV replicativeintermediates were observed by southern blot. The surface antigen and envelop antigen ofHBV were assayed by chemiluminescence immunoassay (CMIA). HBV virus load insupernatant of HepG2215 were detected by real time PCR.4. HepG2215 cells were treated with inhibitors of NF-κB, IRF3, or MAPK kinasepathway. After poly(dA-dT)·poly(dT-dA) stimulation, HBV replicative intermediates were observed by southern blot. Phosphorylated ERK were detected by western blot.5. The expression of DAI(DLM-1/ZBP1) molecule in Hun7, HepG2, and HepG2215 celllines were detected by RT-PCR, real time RT-PCR, and western blot, respectively.DAI(DLM-1/ZBP1) cDNA from PBMC were cloned.6. HepG2215 and HepG2 cells were transfected with DAI(DLM-1/ZBP1) siRNA. Afterpoly(dA-dT)·poly(dT-dA) stimulation, HBV replicative intermediates were observed bysouthern blot, and the expression of IFIT1 were detected by real time RT-PCR.7. HepG2215 cells were transfected with poly(dA-dT)·poly(dT-dA). The expression andtranslocation of high-mobility group box-1 (HMGB1) were detected by western blot.Results1. Poly(dA-dT)·poly(dT-dA) was able to induce typeⅠinterferon in hepatocyte-derivedcells in a time- and dose-dependent manner. However, the kinetic of the IFN-βand IFIT1expression in HepG2215 was delayed and differed strongly from that in HepG2, whichIFN-β/ISG expression peak at 6h and decreased gradually. TNF-αwas induced andmaintained a stable level from 6h to 72h in HepG2.2.15 after poly(dA-dT)·poly(dT-dA)stimulation. In HepG2 cells, TNF-αexpression had no significant difference in compare withnon-treatment group. The cytokine productions by poly(dA-dT)·poly(dT-dA) treatmentinvolved the activation of NF-κB and IRF3.2. Surprisingly, HBV replication was up-regulated in HepG2215 afterpoly(dA-dT)·poly(dT-dA) transfection despite the activation of some ISGs. But there was nodifference of HBV proteins expression and HBV viral loads in the supernatant of HepG2215before and after poly(dA-dT)·poly(dT-dA) transfection. HBV knockdown in HepG2.2.15 orHBV over-expression in HepG2 cells didn't change the kinetics of ISG expression. However,the IFIT1 was up-regulated in HepG2.2.15 cells in response to poly(dA-dT)·poly(dT-dA)stimulation after pretreatment with HBV siRNA.3. HBV replication was not significantly affected by treatment of HepG2215 cells withculture media harvested from cells transfected with different doses ofpoly(dA-dT)·poly(dT-dA) indicating that dsDNA-activated enhanced HBV replication waspredominantly mediated by intracellular factors, rather than secreted cytokines. Preliminaryresults indicated that U0126, an inhibitor of MAPK-ERK1/2, could block thepoly(dA-dT)·poly(dT-dA) enhanced HBV replication. Western blot results showed that phosphorylated ERK1/2 was attenuated in HepG2.2.15 cells after poly(dA-dT)·poly(dT-dA)stimulation. It implys that poly(dA-dT)·poly(dT-dA) could enhance HBV replication throughdecreasing ERK1/2 phosphorylation.4. DAI(DLM-1/ZBP1) could be detected in Huh7, HepG2, and HepG2215 cell lines.Five variants were cloned fron human PBMC including naturally occurring dominantnegative variants. As a first reported cytosolic DNA sensor, DAI(DLM-1/ZBP1) didn't beeninvolved in ISG response to poly(dA-dT)·poly(dT-dA) stimulation in both HepG2 andHepG2.2.15 cells. But the enhanced HBV replicative intermediates induced bypoly(dA-dT)·poly(dT-dA) in HepG2.2.15 cells possibly required DAI(DLM-1/ZBP1)molecule.5. Poly(dA-dT)·poly(dT-dA) transfection had no effect on HMGB1 expression andsecretion in HepG2215 cells. But the expression level of HMGB1 were decreased in bothcytoplasm and nucleus of HepG2 cells after poly(dA-dT)·poly(dT-dA) stimulation. It meansthat ds-DNA can induce HMGB1 secretion from HepG2 cells.Conclusions1. Double-stranded DNA is able to elute innate immune response in hepatocyte-derivedcells and the activation of NFκB and IRF3 are involved in this signaling pathway.2. HBV replication is enhanced in HepG2215 after dsDNA stimulation, which isaccompanied with attenuated pERK.3. These results suggest that more careful consideration should be given whendouble-stranded DNA are used as gene delivery system in HBV-infected disease treatment.