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. IFN-α has been shown to modify cccDNA-bound histones, but it is not known whether the anti-HBV effect of IFN-α involves methylation of cccDNA. The present study aimed to determine whether IFN-α induced methylation of HBV cccDNA in a cell-based model in which HepG2 cells were directly infected with wild-type HBV virions. Methylation status of HBV cccDNA was assessed using global DNA methylation ELISA assay, methylation-specific PCR and bisulfite sequencing. IFN-α suppressed HBV DNA and RNA transcripts, but methylation profiles were similar between the control and IFN-α treated groups. Chromatin immunoprecipitation results revealed binding of DNA methyltransferases (DNMT) 3A and DNMT3B to HBV cccDNA and treatment with IFN-α suppressed the recruitment of DNMT3B to cccDNA. Taken together, these results suggest that IFN-α does not induce methylation of HBV cccDNA. Therefore, it was concluded that methylation is unlikely to contribute to the anti-HBV effect of IFN-α in HepG2 cells, and that alternative mechanisms need to be sought to enhance cccDNA methylation as a novel therapy against HBV.