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.
Highlights
Hepatitis B virus (HBV) causes widespread infection in humans, leading to chronic hepatitis in approximately 400 million people worldwide [1]
HBV genomic relaxed circular (RC) DNA is delivered into the nucleus and converted into the covalently closed circular DNA, which assembles into a viral minichromosome architecture [5,6,7,8]
We demonstrated that while CpG island I is barely methylated, methylation of CpG island II is associated with low HBV DNA serum titer, and methylation of CpG island III may contribute to a lower serum HBsAg level in chronic hepatitis B patients
Summary
Hepatitis B virus (HBV) causes widespread infection in humans, leading to chronic hepatitis in approximately 400 million people worldwide [1]. These patients, if untreated, suffer an elevated risk of liver fibrosis, cirrhosis, and ultimate hepatocellular carcinoma [2,3,4]. HBV is an enveloped double-stranded DNA virus that mainly infects hepatocytes [5]. Nucleocapsids containing mature RC DNA genome are either enveloped by viral surface proteins (HBsAg) and exported as virions, or recycled back to the nucleus to replenish the cccDNA pool [5,9]. HBV infection is maintained by the persistence of nuclear cccDNA episome
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