Methylation regulates hepatitis B viral protein expression.

BACKGROUND Hepatitis B virus (HBV) DNA has been shown to contain CpG islands that are methylated in human tissue, which suggests a role for methylation in regulating viral protein production. However, data are lacking about whether methylation regulates viral gene expression. METHODS To investigate the hypothesis that methylation of viral DNA regulates viral gene expression, unmethylated, partially methylated, and fully methylated viral DNA was transfected into HepG2 cells. In addition, a new assay was designed that specifically identifies methylated covalently closed circular DNA (cccDNA) in human liver tissue. RESULTS Transfection of methylated HBV DNA led to reduced HBV mRNA levels in HepG2 cells, decreased surface and core protein expression in these cells, and decreased secretion of HBV viral proteins into the cell supernatant. These data provide direct evidence that CpG islands regulate gene transcription of HBV. Furthermore, methylated cccDNA was found in tumor and nonneoplastic human liver tissues. Finally, an in vitro equivalent of cccDNA showed decreased viral protein production in HepG2 cells after DNA methylation. CONCLUSION Taken together, these data demonstrate that methylation of viral CpG islands can regulate viral protein production.