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

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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.

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Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

P Jones, G Veenstra, P A Wade … (1998)

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

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IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.

Laura Belloni, Lena Allweiss, Francesca Guerrieri … (2012)

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.

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Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.

Laura Belloni, Teresa Pollicino, Francesca De Nicola … (2009)

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.

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Author and article information

Contributors

Wang-Shick Ryu: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2014

Publication date (Electronic): 22 October 2014

Volume: 9

Issue: 10

Electronic Location Identifier: e110442

Affiliations

[1 ]Department of Infectious Diseases, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China

[2 ]Institute for Biotechnology and Virology Research, Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania, United States of America

[3 ]Key Laboratory of Medical Molecular Virology (MOH & MOE), Shanghai Medical College, Fudan University, Shanghai, China

Yonsei University, Republic Of Korea

Author notes

* E-mail: jmzhang@ 123456fudan.edu.cn (JZ); haitguo@ 123456iupui.edu (HG)

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: Yongmei Zhang RM HG JZ. Performed the experiments: Yongmei Zhang RY DC Yijun Zhang. Analyzed the data: Yongmei Zhang HZ YK. Contributed reagents/materials/analysis tools: HL JW YQ YH. Wrote the paper: Yongmei Zhang HG JZ.

[¤]

Current address: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America

Article

Publisher ID: PONE-D-14-22105

DOI: 10.1371/journal.pone.0110442

PMC ID: 4206413

PubMed ID: 25337821

SO-VID: af6e4a6f-ec5a-4f26-9063-692ade030bba

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 18 May 2014

Date accepted : 12 September 2014

Page count

Pages: 12

Funding

This work was supported by National Natural Science Foundation of China (81071354, 81271833), 973 project (2012CB519001), and National Science and Technology Major Project of China (2012ZX10002007-001-002, 2013ZX10002001) (to Jiming Zhang) and National Institutes of Health (NIH) R01AI094474 (to Haitao Guo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Data Availability The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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

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Most cited references 30

Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.

Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.

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