Preliminary Study On The Effect Of Hepatitis B Virus (HBV) On Interferon Induction In Human Hepatocytes

Hepatitis B virus(HBV) infection has been a serious health problem worldwide. The most significant pemiciousness of HBV infection is its easiness to become persistent infection,which may lead to chronic hepatitis,liver cirrhosis and hepatocarcinoma.To date,the mechanism of chronic HBV infection remains obscure. However,some reports suggested that the evasion of immune surveillance of the host is a predominant strategy for the persistent HBV infection.The interferon system of innate immunity has not only direct antiviral activity but mediates the outset of adaptive immunity,indicating an important role of interferon in controlling virus infection.However,clinical data showed that there are only about 30%-40%of patients with chronic hepatitis B respond persistently to interferon treatment of alpha-interferon alone,suggesting that HBV may have an antagonistic activity to the antiviral activity of interferon.Indeed,it is reported that HBV inhibits the nuclear translocation or methylation of STAT1,an important transcription factor in interferon mediated antiviral activity.There is also a report suggest that the core and pre-core protein of HBV can bind directly to the promoter of Mx,a classical antiviral gene, and down regulate the expression of Mx protein.These clinical data combined with the basic studies,therefore,fully suggest an antagonism between HBV infection and antiviral activity of interferon.However,whether HBV inhibits the production of interferon in human hepatocytes and the underlying mechanism still remains unclear. Although one group reported that the core protein of HBV could inhibit the production of IFN-βin vitro,it is still not conformed in hepatocytes in vivo and the mechanism awaits further exploration.In light of the report that human primary hepatocytes are interferon competent cells,it is an urgent need to examine whether and how HBV affects the production of interferon in human hepatocytes.To study the effect of HBV on the production of interferon in human hepatocytes,we firstly examined and compared the competency of interferon induction of several different liver derived cells.Two hepatoma cell lines,Huh7 and HepG2,and one primary human hepatocyte cell line PH5CH8 were treated with Newcastle disease virus(NDV),a potent inducer of interferon production,and Real-time RT PCR result showed that while Huh7 and HepG2 cells could hardly be induced to produce IFN-β,PH5CH8 cells produces large amounts of IFN-βafter induction by NDV,suggesting that primary human hepatocytes are strong interferon producers and the PH5CH8 cells can be used as a cell model for our further study. To learn whether HBV replication can inhibit the production of interferon in human hepatocytes,the PH5CH8 cells were transfected with a HBV-replicating plasmid pHBV3.8 and then were treated w:ith NDV.RT-PCR result showed that the cells transfected with pHBV3.8 and induced by NDV produced.significantly lower amount of IFN-βthan the control group,suggesting that HBV replication has an inhibitory effect on the interferon production in PH5CH8 cells.To further confirm the effect of HBV on the production of interferon in human hepatocytes,PH5CH8 cells were tansfected with HBV-replicating plasmid pCMV-HBV and replication-abrogated plasmid pHBV-YMDD,Real-time RT PCR result suggested that,as compared with control,the cells transfected with pCMV-HBV produced much less IFN-β,while transfection of pHBV-YMDD had no effect,indicating that HBV replication inhibits IFN-βproduction in PH5CH8 cells.To further determine the effect of HBV proteins on interferon production,PH5CH8 cells were tansfected with a reporter plazmid of IFN-βpromoter plFN-β-1uc,Rluc and plasmids expressing HBV proteins including polymerase,core and HBx.The result of dual luciferase assay showed that,compared with the control,the luciferase activity of IFN-βpromoter in the cells transfected with polymerase was significantly lower, while there is no significant difference in the groups that were transfected with core and HBx,suggesting that HBV polymerase have a strong inhibitory effect on the IFN-βpromoter activity in human hepatocyte cell line PH5CH8.To conform the effect of HBV proteins on interferon production,the plasmids expressing HBV proteins were transfected into Huh7 cells and then were tranfected with Poly(I:C). Real-time RT PCR result showed that the mRNA level of IFN-βtranscribed from the cells transfected with polymerase expressing plasmid was significantly lower than the control group,while transfection of core and HBx expressing plasmid had no significant difference,suggesting that HBV polymerase inhibits interferon production also in Huh7 cells.Moreover,the tranfection of different amounts of plasmids expressing polymerase showed an inhibitory effect on both IFN-βpromoter activity and mRNA level in a dose dependent manner in PH5CH8 cells.Therefore,these data suggest that HBV polymerase may be a viral regulator negatively modulating the interferon production pathway during HBV infection in human hepatocytes.To identify the region that accounts for the inhibitory effect of polymerase on interferon production in human hepatocytes,different truncated mutants of polymerase were transfacted into PH5CH8 cells,dual luciferase assay result suggested that the mutants containing N-terminal 691 amino acid,347 amino acid and 178 amino acid has a significant inhibition on the IFN-βpromoter activity while the TP depleted mutant has much weaker inhibitory effect,suggesting that the TP domain of HBV polymerase is crucial for the inhibition of interferon production by HBV polymerase in human hepatocytes.Next,we want to identify which site of the interferon induction pathway was inhibited by HBV.Given the fact that Poly(I:C) and NDV induced signaling converges at the activation of IRF-3 by TBK1/IKKε,therefore,pHBV3.8,pIFN-β-luc, Rluc,IRF-3 and TBK1 or IKKεwere transfected into Huh7 cells to analyze the effect of HBV on TBK1/IKKεactivated signaling.The result of dual luciferase assay suggested that HBV inhibits significantly the IFN-βluciferase activity activated by both TBK1 and IKKε,indicating that HBV inhibits interferon induction by interfering with the signaling downstream TBK1 and IKKε.In light of the central role of IRF-3 activation in the interferon indcution pathway,we next want to identify whether phosphorylation of IRF-3 was inhibited by HBV and its proteins.Therefore,pHBV3.8 as well as plsamids expressing HBV proteins were transfected into PH5CH8 cells and the result of Western blot analysis using an antibody specifically against phospho-Ser-396 IRF-3 showed that there is no significant difference of IRF-3 phosphorylation status between the positive control and the groups transfected with HBV and its protein expressing plasmids,suggesting that inhibition of interferon induction by HBV is not through interfering the activation of IRF-3.Taken together,this study suggests that HBV can inhibit induction of interferon in human hepatocytes,which may imply another important strategy for evasion of immune surveillance of the host by HBV.Besides,our data also suggest that HBV polymerase may play a critical role in the inhibition of interferon production in human hepatocytes by HBV and this inhibition is through interfering the signaling activated by TBK1/IKKε.Further analysis of the mechanism underlying the inhibition of interferon induction by HBV polymerase in human hepatocytes will shed light on the understanding of strategy of immune evasion of HBV and its persistent infection and may also provide new insights into developing new approaches for treatment of chronic hepatitis B.