Functional Role Of Dicoumarol On HBV Replication And Transcription Via Regulating HBx Protein Stability

Objective1.To investigate the regulatory role of NQO1 inhibitor dicoumarol（DIC）in HBV transcription and replication.2.To study the molecular mechanism of NQO1 in maintaining HBx protein stability.3.To clarify the important role of NQO1 stabilized HBx protein on cccDNA transcription and HBV replication.4.To explore the molecular mechanism of NQO1/HBx in cccDNA transcription.5.To evaluate the anti-HBV efficacy of NQO1 inhibitor DIC in vivo.Methods1.The inhibitory effect of NQO1 inhibitor DIC on HBx protein expression were detected by Nano-Glo^?HiBiT Lytic Detection System and western blot.2.The HBV-infected HepG2-NTCP cells were exposed to a series concentration of DIC.The effect of DIC on HBV RNAs were determined by real-time PCR（RT-PCR）and northern blot.Moreover,the HBV cccDNA level in DIC-treated cells were determined by quantitative real-time PCR（qRT-PCR）.3.The NQO1 knock down/out or overexpression cells were cotransfected with HBx:the mRNA and protein level were measured by RT-PCR and western blot,respectively;those cells were further treated with CHX to measure the half-life of HBx by western blot.4.The NQO1 knock down/out or overexpression cells were cotransfected with HBx and treated with MG132 followed by western blot to examined the level of HBx.Furthermore,Huh-7 cells were cotransfected with HBx and HA-Ubiquitin,HBx ubiquitination was examined by western blot.In addition,Huh-7 cells were transfected with HBx and the combination among HBx,NQO1 and 20S proteasome were examined by co-immunoprecipation.5.HBV infected cells（HepG2-NTCP and PHH）were transduction with lenti-virus expression NQO1 or NQO1^mut.The effect of NQO1 on HBV RNA were determined by RT-PCR and northern blot.Moreover,the HBV cccDNA level in NQO1 overexpression cells were determined by qRT-PCR.6.HBV infected HepG2-NTCP cells were transduction with lenti-virus expression NQO1 and treated with EU followed by RT-PCR to determine the newly synthesized EU-labeled total HBV RNAs and pgRNA.Moreover,cells were exposed to 7-ADD and the total HBV RNAs and pgRNA were quantified by RT-PCR.7.HBV infected cells（HepG2-NTCP and PHH）were transduction with lenti-virus expression shNQO1.The effect of NQO1 knock down on HBV RNA were determined by RT-PCR and northern blot.In addition,the HBV cccDNA level in NQO1 overexpression cells were determined by qRT-PCR.8.HepG2-NTCP cells were infected with wild type（HBV-WT）or HBx protein deficient virus（HBV-ΔHBx）and then were transduced with lentivirus expressing NQO1 or shNQO1.Total HBV RNAs and pgRNA were determined by RT-PCR.Also,cccDNA level were detected by qRT-PCR.9.HBV infected HepG2-NTCP cells were transduced with lentivirus expressing NQO1 or shNQO1 or treated with DIC,the combination between HBx and cccDNA were evaluated by ChIP assay.10.HBV infected HepG2-NTCP cells were transduced with lentivirus expressing NQO1 or shNQO1 or treated with DIC,the level of H3K9ac,H3K4me3,H3K9me3 and H3K27me3 associated with cccDNA were analyzed by ChIP assay.11.HBV infected mice were treated with DIC or ETV.The serum level of HBsAg and HBV DNA were determined by ELISA and qRT-PCR,respectively.Next,intrahepatic total HBV RNAs and HBV 3.5-kb RNA were detected by RT-PCR;cccDNA level were measured by qRT-PCR;protein level of HBx and HBs were examined by IHC.Importantly,the level of HBx,H3K9ac,H3K4me3,H3K9me3 and H3K27me3 associated with cccDNA were analyzed by ChIP assayResults1.DIC reduced the levels of HiBiT-HBx in a dose-dependent manner.Moreover,treatment with increasing concentrations of DIC resulted in dose-dependent decrease exogenous HBx in HCC cells2.DIC reduced the levels of both HBV total RNAs and pgRNA in a dose dependent manner,without affecting cccDNA level。3.NQO1 knock down/out significantly decreased HBx expression in HepG2 cells ectopically expressing HBx,whereas no effect was observed on HBx mRNA level.In contrast,NQO1 overexpression increased HBx protein level without affecting its mRNA level.CHX chase assay showed that the half-life of HBx protein was markedly decreased in NQO1 knock down/out cells,while increased in NQO1 overexpression cells.4.The effect of NQO1 knock down/out or overexpression on HBx was blocked in the presence of MG132.Also,ubiquitinated-HBx level remained unchanged in cells.Furthermore,HBx and NQO1 were physically associated with 20S proteasome.5.Expression of exogenous NQO1,but not NQO1Y^127/129A enhanced total HBV RNAs,pgRNA and HBV DNA levels in HBV-infected cells（HepG2-NTCP and PHH）,without affecting cccDNA levels.Notably,cccDNA transcription activity（Total RNA/cccDNA and pgRNA/cccDNA）were increased in NQO1 overexpression cells.6.Nascent RNA capture assays showed that the rate of HBV total RNAs and pgRNA synthesis were increased in HBV-infected cells ectopically expressing NQO1.Meanwhile,the half-life of HBV total RNAs and pgRNA was unaffected by NQO1 after actinomycin D treatment.7.NQO1 knock down reduced total HBV RNAs,pgRNA and HBV DNA levels in HBV-infected cells（HepG2-NTCP and PHH）,without affecting cccDNA levels.Notably,cccDNA transcription activity（Total RNA/cccDNA and pgRNA/cccDNA）were decreased in NQO1 knock down cells.8.Total HBV RNAs,pgRNA level and cccDNA transcription activity（Total RNA/cccDNA and pgRNA/cccDNA）were elevated by NQO1overexpression in HepG2-NTCP cells infected with wild type virus（HBV-WT）,while decreased by NQO1 knock down.However,HBx deficiency abrogated the enhancement of HBV RNAs mediated by NQO1or decrease of HBV RNAs induced NQO1 knock down.9.The combination of HBx to cccDNA were enhanced by NQO1overexpression,while reduced by NQO1 knock down or DIC treatment.10.The cccDNA ChIP assay demonstrated that NQO1 knock down or DIC treatment resulted in a decrease of active histone marks（H3K9ac and H3K4me3）and an increase of repressive modifications（H3K9me3 and H3K27me3）.However,ectopic expression of NQO1 resulted in opposite effect on cccDNA.11.The in vivo data displayed that DIC treatment markedly reduced serum HBsAg and HBV DNA level.Furthermore,DIC treatment reduced intrahepatic total HBV RNAs and HBV 3.5-kb RNA,as well as protein level of HBx and HBs.cccDNA-ChIP assay illustrated that DIC treatment resulted in decreased level of HBx,H3K9ac and H3K4me3 but increased level of H3K9me3 and H3K27me3 on cccDNA.ConclusionIn this study,we found that the NQO1 inhibitor DIC could significantly inhibit the expression of the viral protein HBx,thereby inhibiting HBV transcription and replication.Further,NQO1 could bind to and protect HBx protein from 20S proteasome-mediated degradation to enhance HBV transcription and replication.Mechanistic study revealed that NQO1 could inhibit HBx degradation to enhance cccDNA transcription activity which was correlates with the increasing active modifications and decreasing repressive modifications on histone.In vivo,DIC showed potent antiviral activity on transcription and replication.In summary,this study analyzed the molecular mechanism of NQO1 regulation of HBx protein stability,clarified the important role of NQO1 stabilized HBx protein on cccDNA transcription and replication,and fully evaluated the antiviral efficacy of NQO1 inhibitor DIC through in vivo and in vitro experiments.