Methylation profile of hepatitis B virus is not influenced by interferon α in human liver cancer cells.

Interferon (IFN) α is used for the treatment of chronic hepatitis B virus (HBV) infection, but the molecular mechanisms underlying its antiviral effect have not been fully elucidated. Epigenetic modifications regulate the transcriptional activity of covalently closed circular DNA (cccDNA) in cells with chronic HBV infection. IFN­α has been shown to modify cccDNA­bound histones, but it is not known whether the anti­HBV effect of IFN­α involves methylation of cccDNA. The present study aimed to determine whether IFN­α induced methylation of HBV cccDNA in a cell­based model in which HepG2 cells were directly infected with wild­type HBV virions. Methylation status of HBV cccDNA was assessed using global DNA methylation ELISA assay, methylation­specific PCR and bisulfite sequencing. IFN­α suppressed HBV DNA and RNA transcripts, but methylation profiles were similar between the control and IFN­α treated groups. Chromatin immunoprecipitation results revealed binding of DNA methyltransferases (DNMT) 3A and DNMT3B to HBV cccDNA and treatment with IFN­α suppressed the recruitment of DNMT3B to cccDNA. Taken together, these results suggest that IFN­α does not induce methylation of HBV cccDNA. Therefore, it was concluded that methylation is unlikely to contribute to the anti­HBV effect of IFN­α in HepG2 cells, and that alternative mechanisms need to be sought to enhance cccDNA methylation as a novel therapy against HBV.

MicroRNA­20 induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells.

Methylation was suggested to suppress the transcriptional activity of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in hepatocytes. This may be associated with its low replicative activity during the inactive stage of chronic HBV infection; however, the exact mechanisms of methylation in HBV infection remain unknown. We have previously shown that short hairpin RNAs induced the methylation of the HBV genome in hepatoma cell lines. We also reported that the microRNA (miR) 17­92 cluster negatively regulates HBV replication in human hepatoma cells. In addition, miR­20a, a member of the miR 17­92 cluster, has sequence homology with the short hairpin RNA that induces HBV methylation. In the present study, we investigated whether miR­20a can function as an endogenous effector of HBV DNA methylation. The results indicated that overexpression of miR­20a could suppress the replicative activity of HBV and increased the degree of methylation of HBV cccDNA in the HepAD38 hepatoma cell line. Argonaute (AGO)1 and AGO2, effectors of the RNA­induced silencing complex, were detected in the nucleus of HepAD38 cells; however, only AGO2 was bound to HBV cccDNA. In addition, intranuclear AGO2 was determined to be bound with miR­20a. In conclusion, miR­20a may be loaded onto AGO2, prior to its translocation into the nucleus, inducing the methylation of HBV DNA in human hepatoma cells, leading to the suppression of HBV replication.