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

Abstract

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.

HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state

“…15,16 DNA hypermethylation of CpG island 2 can repress transcription and replication of HBV. [2][3][4] We found that the relative abundances of HBc binding to CpG island 2 were negatively correlated with the levels of DNA methylation at CpG island 2. These data indicated that binding of HBc to CpG island 2 inhibited DNA methylation.…”

“…1 Recent advances have highlighted the epigenetic mechanisms underlying the regulation of HBV cccDNA expression. [2][3][4][5][6] Previous studies have found that hypermethylation of CpG island 2 is associated with an impaired replication of intra-hepatic cccDNA in patients with chronic Hepatitis B (CHB). [2][3][4] Furthermore, hypermethylation of CpG islands is observed in individuals with occult HBV infection.…”

“…[2][3][4][5][6] Previous studies have found that hypermethylation of CpG island 2 is associated with an impaired replication of intra-hepatic cccDNA in patients with chronic Hepatitis B (CHB). [2][3][4] Furthermore, hypermethylation of CpG islands is observed in individuals with occult HBV infection. 3,4 Additionally, acetyltransferase CREB binding protein (CBP) and histone deacetylase 1 (HDAC1) can be recruited to minichromosomes, and they promote the hypoacetylation of cccDNA-bound H3/H4 histones, leading to the inhibition of cccDNA transcription.…”

Viral epigenomes in human tumorigenesis

“…All studies, from the first in 1983 (Miller and Robinson, 1983) to those of the present day, have revealed DNA methylation in the HBV genome. In addition, DNA methylation of several viral genes is known to be associated with loss of their expression (Vivekanandan et al, 2008(Vivekanandan et al, , 2009Fernandez et al, 2009;Guo et al, 2009). Nevertheless, very few studies have attempted to analyze the viral methylation of specific sequences.…”

Current and future antiviral drug therapies of hepatitis B chronic infection

“…In CHB patient, viral and host DNA methylation density varies significantly has been identified as a host defence mechanism to suppress viral gene expression and replication. Furthermore, an up regulation of DNA methyltransferases has been reported in CHB livers that facilitates the methylation of cccDNA and viral genomes affecting protein production and viral replication [74,75] . It has been reported that host DNA methylation is the main mechanism to inactivate relevant genes in HCC [76] .…”