Transcription of Hepatitis B Virus Covalently Closed Circular DNA Is Regulated by CpG Methylation during Chronic Infection

Abstract

The persistence of hepatitis B virus (HBV) infection is maintained by the nuclear viral covalently closed circular DNA (cccDNA), which serves as transcription template for viral mRNAs. Previous studies suggested that cccDNA contains methylation-prone CpG islands, and that the minichromosome structure of cccDNA is epigenetically regulated by DNA methylation. However, the regulatory effect of each CpG island methylation on cccDNA activity remains elusive. In the present study, we analyzed the distribution of CpG methylation within cccDNA in patient samples and investigated the impact of CpG island methylation on cccDNA-driven virus replication. Our study revealed the following observations: 1) Bisulfite sequencing of cccDNA from chronic hepatitis B patients indicated that CpG island I was seldom methylated, 2) CpG island II methylation was correlated to the low level of serum HBV DNA in patients, and in vitro methylation studies confirmed that CpG island II methylation markedly reduced cccDNA transcription and subsequent viral core DNA replication, 3) CpG island III methylation was associated with low serum HBsAg titers, and 4) Furthermore, we found that HBV genotype, HBeAg positivity, and patient age and liver fibrosis stage were also relevant to cccDNA CpG methylation status. Therefore, we clearly demonstrated that the status of cccDNA methylation is connected to the biological behavior of HBV. Taken together, our study provides a complete profile of CpG island methylation within HBV cccDNA and new insights for the function of CpG methylation in regulating HBV cccDNA transcription.

Core protein: A pleiotropic keystone in the HBV lifecycle

“…The presence of Cp on CpG island 2 correlates with increased activity of cccDNA (i.e. viral load), while methylation of DNA in CpG islands 2 and 3 correlates with decreased cccDNA activity (Guo et al, 2011; Zhang et al, 2014). Of note, small molecules that drive Cp assembly (Bourne et al, 2008), presumably depleting the concentration of free Cp, led to depletion of cccDNA-bound Cp and decreased production of viral RNA and proteins (Belloni et al, 2013).…”

Persistent Loss of Hepatitis B Virus Markers in Serum without Cellular Immunity by Combination of Peginterferon and Entecavir Therapy in Humanized Mice

“…The CpG islands were defined based on the following criteria: a GC content of Ն50%, an observed-to-expected CpG dinucleotide ratio of Ն0.60, and a sequence window longer than 100 bp. Bisulfite sequencing primers were designed separately for both CpG islands II (nt 1228 to 1663) and III (nt 2295 to 2446) to avoid overlap between CpG sites (Table 1) (32). DNA samples were modified using the Cells-to-CpG bisulfite conversion kit (Applied Biosystems) according to the manufacturer's specifications.…”

“…Island I contains the start site of the S gene, island II spans a region that overlaps enhancer I/II and is proximal to the core promoter, and island III covers the start codon of the polymerase gene and the upstream region of the SP1 promoter (32,33). It was reported that transcription of HBV cccDNA is regulated by CpG methylation during chronic infection (27,33).…”

Current and future directions for treating hepatitis B virus infection

“…In the HBV-infected liver, free HBV DNA and its products are causally related to the activity of liver disease, but the persistence of HBV infection is maintained by the nuclear cccDNA, which serves as a transcription template for HBV mRNA [111,112] . Although there are several opposing views [113] , it was reported that HBV cccDNA is noncytolytically degraded by agents that upregulate apolipoprotein B mRNA editing enzyme and catalytic polypeptide-like (APOBEC) 3A and 3B [23] .…”