Additional file 1 of Assembly and comparative analysis of the complete mitochondrial genome of Viburnum chinshanense

Additional file 1: Figure S1. The full alignment of Sanger sequencing reads. p1, p2, p3, and p4 represent path 1, path 2, path 3 and path 4 respectively

Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection

<div><p>Chronic hepatitis B virus (HBV) infection is associated with functionally impaired virus-specific T cell responses. Although the myeloid-derived suppressor cells (MDSCs) are known to play a critical role in impairing antiviral T cell responses, viral factors responsible for the expansion of MDSCs in chronic hepatitis B (CHB) remain obscure. In order to elucidate the mechanism of monocytic MDSCs (mMDSCs) expansion and T cell function suppression during persistent HBV infection, we analyzed the circulation frequency of mMDSCs in 164 CHB patients and 70 healthy donors, and found that the proportion of mMDSCs in HBeAg (+) CHB patients was significantly increased compared to that in HBeAg (-) patients, which positively correlated with the level of HBeAg. Furthermore, exposure of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to HBeAg led to mMDSCs expansion and significant upregulation of IL-1β, IL-6 and indoleamine-2, 3-dioxygenase (IDO), and depletion of the cytokines abrogated HBeAg-induced mMDSCs expansion. Moreover, HBeAg-induced mMDSCs suppressed the autologous T-cell proliferation <i>in vitro</i>, and the purified mMDSCs from HBeAg (+) subjects markedly reduced the proliferation of CD4⁺ and CD8⁺ T cells and IFN-γ production, which could be efficiently restored by inhibiting IDO. In summary, HBeAg-induced mMDSCs expansion impairs T cell function through IDO pathway and favors the establishment of a persistent HBV infection, suggesting a mechanism behind the development of HBeAg-induced immune tolerance.</p></div

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

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

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

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

Core protein is not required for HBV transcription in the monomeric linear HBV genome transfection assay.

<p>HepG2 cells were seeded into 6-well-plate and transfected with 4 µg of monomeric linear full-length wild type or core-null HBV DNA for 5 days. One set of wild type HBV linear DNA transfected cells were treated with HBV reverse transcriptase inhibitor Lamivudine (2′,3′-dideoxy-3′-thiacytidine, 3TC) at the concentration of 10 µM. (A) Southern blot analysis of HBV protein-free Hirt DNA recovered from the transfected cells. One set of samples were digested with DpnI before gel loading. Full length linear HBV DNA served as 3.2 kb size marker. In vitro ligation products of linear HBV DNA by T4 DNA ligase served as 3.2 kb DNA ladder. (B) Intracellular HBV RNA and core DNA were analyzed by Northern and Southern blot hybridization, respectively. For RNA analysis, 5 µg of total RNA was loaded on each lane, and ribosomal RNA (28S and 18S) was used as loading controls. The positions of HBV RNA species are marked. For core DNA analysis, half volume of the DNA samples was subjected to electrophoresis. The positions of HBV RC DNA and SS DNA are labeled.</p

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

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