Evidence that methylation of hepatitis B virus covalently closed circular DNA in liver tissues of patients with chronic hepatitis B modulates HBV replication

Epigenetic factors may modulate chronic Hepatitis B viral infection by affecting virion gene transcription. The aim of this study was to compare the methylation status of the intrahepatic covalently closed circular DNA (cccDNA) CpG island 2 and HBV replication capability. HBV cccDNA was extracted from liver biopsies of 55 HBsAg‐positive patients with chronic hepatitis B (32 HBeAg‐positive and 23 HBeAg‐negative), and was analyzed for methylation status and quantity. The two Hpa II recognition sequences CCpGG in the CpG island 2 were methylated in infected liver tissues from 24 (43.6%) of 55 patients. Positive ratios of cccDNA methylation were significantly higher in HBeAg‐negative patients (15/23, 65.2%) than HBeAg‐positive patients (9/32, 28.1%) ( P  < 0.05). The percentage of methylated‐cccDNA/total‐cccDNA of HBeAg‐negative samples (a median of 48%, ranging from 5% to 83%) was significantly higher ( P  < 0.001) than HBeAg‐positive samples (a median of 14%, ranging from 0.26% to 35%). Ratios of relaxed circular DNA (rcDNA) to cccDNA molecules revealed that cccDNA methylation correlated with impaired virion productivity in HBeAg‐positive individuals ( P  < 0.05). The bisulfite DNA sequencing showed that methylation density was significantly higher in HBeAg‐negative than in HBeAg‐positive patients ( P  < 0.05). The methylation level of the CpG island 2 of the cccDNA in HBeAg‐negative patients was higher than that in HBeAg‐positive patients, suggesting that HBV cccDNA methylation may be relevant to replication capability of HBV. J. Med. Virol. 81:1177–1183, 2009. © 2009 Wiley‐Liss, Inc.