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Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin conformation amenable to epigenetic manipulation

ABSTRACT: Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative since it does not eliminate nuclear HBV cccDNA, the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target the transcriptional regulation of cccDNA would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes and from HBV infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome, and surprisingly very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68402 | GEO | 2015/10/08

SECONDARY ACCESSION(S): PRJNA282651

REPOSITORIES: GEO

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Hepatitis B virus core protein is not required for cccDNA transcriptional regulation

Project description:Background & Aims: Hepatitis B virus (HBV) infection is a major health burden worldwide and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment. HBV core protein (HBc) has merged as a promising antiviral target, as it plays important roles in critical steps of viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains to be illustrated. Methods: Synthesized HBV cccDNA and HBVcircle with or without HBc deficiency were transfected into hepatocytes. A recently reported Adeno-Associated Virus (AAV) mediated HBV cccDNA mouse model was employed. Two capsid assembly modulators (CAMs) were used. HBV replication markers were evaluated. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays were conducted with different transcription factors, histones and RNA polymerase 2. Results: In HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen production. Reconstitution of HBc did not change cccDNA derived HBV markers. Similar results were obtained in vivo, from mouse cccDNA model. ChIP data revealed similar transcription regulation of HBc deficient cccDNA chromatin with wide type cccDNA. Furthermore, CAMs treatment could not alter cccDNA transcription. Conclusions: Our results indicate that HBc neither affects histone modifications and transcription factors binding of cccDNA, nor influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, it does not suppress cccDNA transcriptional activity. Thus, therapeutic targeting capsid or HBc is not sufficient to reduce cccDNA transcription.

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CTCF regulates hepatitis B virus cccDNA chromatin topology [RNA-seq]

Project description:Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcripts, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessibility/sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down- stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

2024-01-18 | GSE249278 | GEO

CTCF regulates hepatitis B virus cccDNA chromatin topology [ATAC-seq]

2024-01-18 | GSE248738 | GEO

Transcription of HBV cccDNA is synchronized at single-cell level and orchestrated by expression of HBx

Project description:Chronic infection of Hepatitis B virus (HBV) remains a public health problem worldwide. HBV infection relies on the persistence of covalently closed circular DNA (cccDNA) in the nucleus and actively cccDNA transcription. To understand HBV cccDNA transcription regulation at single cell level, we isolated primary human hepatocytes from liver humanized FRG mice infected by one or more (two or three) HBV genotypes, and we quantified transcripts of HBV structural genes in single cells. HBV transcripts were ascribed to the transcription of individual HBV genes by 5’ end sequencing thus avoiding the ambiguity caused by the overlap of viral genome coding at the 3’ ends. Transcripts from different cccDNA in single cells were separated according to the single-nucleotide polymorphism (SNP) among different HBV genotypes. We found that the transcription of HBV follows “all-or-none” pattern in single cells: either all of the individual cccDNA molecules actively transcribe simultaneously, or, none of them generates transcripts of the structural genes. In vitro cell infection assays with recombinant HBV are consistent with the sequencing results of ex vivo samples from natural HBV infection, and also confirm that such a pattern is apparently controlled by the expression of HBx protein. These results strongly support a synchronized transcription model of HBV cccDNA molecules in single hepatocytes, and provide new insight helpful for developing HBV cure strategy.

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Binding of Nucleolin to HBV cccDNA

Project description:Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. In this study we sought to identify host factors interacting with the HBV genome. Nucleolin (Ncl) was identified as a potential interactor of HBV cccDNA. This interaction was veriefied using an established ChIPseq protocol. The data show that Ncl binds cccDNA albeit at lower levels than HBcAg. Ncl deposition occurs across the genome without a clear localization and a variable pattern of deposition between experiments. This verifies the interaction of Ncl with HBV-cccDNA.

2021-12-31 | GSE123715 | GEO

Interferon-induced degradation of the hepatitis B virus persistence form, cccDNA, depends on ISG20

Project description:Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Cytokine treatments can diminish HBV cccDNA via APOBEC3-mediated deamination. Here we show that overexpression of APOBEC3A alone, however, was not sufficient to reduce cccDNA in HBV-infected cells. This required addition of interferon indicating that cccDNA degradation requires an additional, interferon-stimulated gene (ISG). Transcriptome analyses identified ISG20 as the only type I and II interferon-induced, nucleus-resident protein with annotated nuclease activity. ISG20 expression was detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion abolished the interferon-induced loss of cccDNA, and co-expression of ISG20 and APOBEC3A was sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires induction of a deaminase and nuclease. Our findings highlight that ISGs cooperate for their antiviral function and this cooperativity may be explored for therapeutic targeting.

2021-04-22 | PXD019285 | Pride

Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx

Project description:In hepatocyte nuclei, hepatitis B virus (HBV) genomes occur episomally as covalently closed circular DNA (cccDNA). The HBV X protein (HBx) is required to initiate and maintain HBV replication and acts as host gene trans-regulator. However, functionally relevant spatiotemporal localization and interactions of cccDNA and HBx remain to be understood. This is the first study utilizing circularized chromosome conformation capture (4C) to identify regional virus-host genome interactions. We combined 4C with RNA-seq and ChIP-seq to illuminate the nuclear landscape associated with HBV episomes and HBx. Moreover, we functionally studied HBx-binding to episomal HBV DNA. In human HBV-positive HepaRG hepatocytes, 4C and ChIP revealed specific nuclear localization of HBV episomes and HBx associated with actively transcribed nuclear domains correlating well in size with constrained topological units built up by chromatin fibre loops, which are believed to constitute fundamental cell nuclear architectural units. Interestingly, HBx alone occupied transcribed chromatin domains, and its binding to HBV episomes depended on its C-terminus in vitro. In conclusion, HBx and HBV DNA similarly follow higher-order nuclear assembly patterns, specifically favoring transcriptionally active nuclear compartments. These novel observations may shed light on important unsolved problems: to understand the long-term episomal stability and the facilitation of viral persistence.

2018-04-30 | GSE100400 | GEO

Identification of cellular protein interacting with HBV genome by protein mass spectrometry

Project description:: Hepatitis B virus (HBV) remains a major public health threat with more than 296 million people chronically infected worldwide at high risk to develop hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. The fundamental limitation rests on the fact that current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. HBV core protein (HBc) is associated with cccDNA. We used an anti-HBc antibody to pull down HBcAg-associated cccDNA complex in nuclear extract of HBV-infected HepG2-NTCP cells. By protein mass spectrometry, we identified many host proteins, including previously known proteins, that are associated with HBV cccDNA. Some of these proteins were functionally validated to play a role in HBV cccDNA activities.

2024-06-16 | PXD042733 | Pride

Mapping the heterogeneity of histone modifications on hepatitis B virus-DNA using liver needle biopsies obtained from chronically infected patients

Project description:Covalently closed circular DNA (cccDNA) forms the basis for replication and persistence of hepatitis B virus (HBV) in the chronically infected liver. We have previously shown through the analysis of de novo HBV infected cell lines that viral transcription is subject to regulation by posttranslational modifications (PTMs) of histone proteins bound to cccDNA. We now report the successful adaptation of this ChIPseq approach for the analysis of fine-needle patient liver biopsy specimens to investigate the role of histone PTMs in chronically HBV-infected patients. Using 18 specimens from patients in different stages of chronic HBV infection our work shows that the profile of histone PTMs in chronic infection is more nuanced than observed in our previous work largely focused on acute infection in in vitro models. Specifically, we find that the majority of recovered HBV sequences are associated with the activating histone PTM H3K4me3, in line with our previous findings. We further find that the striking interpatient variability of its deposition in this patient cohort is linked to viral transcription and patient HBeAg status. Unexpectedly, we detect a significant localized deposition of the inhibitory histone PTM H3K9me3 on HBV-DNA in select patient biopsies which was not observed previously. Altogether, our results show that current in vitro models of HBV infection are unable to fully recapitulate the complex epigenetic landscape of chronic HBV infection observed in vivo and demonstrate that fine needle liver biopsy specimens can provide sufficient material to further investigate the interaction of viral and host proteins on HBV-DNA.

2019-02-07 | GSE113879 | GEO

Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.

Project description:Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.

| S-EPMC4620859 | biostudies-literature

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