Comparative Analysis of CpG Islands among HBV Genotypes

<div><p>DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.</p> </div

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

<div><p>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.</p></div

HBV CpG methylation is associated with patient age and liver fibrosis stage.

<p>(A) CpG II methylation density differed significantly between patients with Knodell fibrosis stage S0-2 and S3-4. (B) Methylation of CpG III was markedly higher in patients with Knodell fibrosis stage S3-4 compared with those with fibrosis stage S0-2. (C) Patients with age ≥40 had higher density of CpG II methylation than and those with age <40.</p

Primer used for hepatitis B virus DNA amplification.

<p>Degenerate base: D (A, T); H (A, T, C); Y (C, T).</p><p>Primer used for hepatitis B virus DNA amplification.</p

Characteristics of the patients and methylation of HBV cccDNA in human liver sample.

<p>Numbers in brackets represent the number of total TA clones; ND: not detected.</p><p>Characteristics of the patients and methylation of HBV cccDNA in human liver sample.</p

The location and size of the three conventional and novel CpG islands within HBV sequences of different genotypes.

<p>–, CpG island was absent. The first T of the <i>Eco</i>RI cleavage site is position 1. The numbering of the nucleotides was determined using an alignment of representative sequences of HBVs of the ten genotypes obtained from GenBank.</p

The genome size, number and mean percentage of nucleotide sequence divergence between HBV genome from genotype A to J analyzed in this study.

<p>The mean percentage of nucleotide sequence divergence between HBV genotype A to J sequences is shown. The corresponding standard deviations of the mean values are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). Analyses were conducted using the Kimura 2-parameter model. The analysis involved 176 nucleotide sequences.</p

The CpG island distribution within representative HBV sequences of HBV genotypes A–J.

<p>The vertical axis refers to the GC percentage, while the horizontal axis refers to the HBV nucleotide sequence. The blue areas represent CpG islands I, II, and III. The vertical red lines indicate CpG dinucleotides.</p

The three novel CpG islands identified in the HBV genome.

<p>The open reading frames of the pre-core/core, polymerase, surface antigen, and X genes are indicated as blue arrows. The four promoters, cp, sp1, sp2, and xp, and main regulatory elements, enhancers I and II (EnhI and EnhII), are indicated as green boxes. The purple areas represent CpG islands I to VI within the HBV genome. Each vertical purple line below indicates a single CpG dinucleotide. The nucleotide position is labeled according to the consensus sequence of HBV genomes with genotype C (EMBL: Y18855–Y18858).</p

Dendrogram based on 176 complete genomic sequences of HBV with genotypes A–J.

<p>The phylogenetic tree was constructed using the UPGMA method. The genotypes are marked by the letters A through J. Each sequence is identified by the GeneBank accession number, followed by the genotype/subgenotype and the country of origin of the isolates. HBV genomes containing three, two, and four CpG islands are shown in blue, red, and green, respectively.</p