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Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.

ABSTRACT: Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a "second-generation" cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.

SUBMITTER: Cai D

PROVIDER: S-EPMC5060949 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.

Cai Dawei D Wang Xiaohe X Yan Ran R Mao Richeng R Liu Yuanjie Y Ji Changhua C Cuconati Andrea A Guo Haitao H

Antiviral research 20160513

Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have prev ...[more]

PMID: 27185623

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Project description:BackgroundHepatitis B virus covalently closed circular DNA (HBV cccDNA) is assembled by histones and non-histones into a chromatin-like cccDNA minichromosome in the nucleus. The cellular histone acetyltransferase GCN5, displaying succinyltransferase activity, is recruited onto cccDNA to modulate HBV transcription in cells. Clinically, IFN-α is able to repress cccDNA. However, the underlying mechanism of IFN-α in the depression of cccDNA mediated by GCN5 is poorly understood. Here, we explored the effect of IFN-α on GCN5-mediated succinylation in the epigenetic regulation of HBV cccDNA minichromosome.ResultsSuccinylation modification of the cccDNA minichromosome has been observed in HBV-infected human liver-chimeric mice and HBV-expressing cell lines. Moreover, histone H3K79 succinylation by GCN5 was identified in the system. Interestingly, the mutant of histone H3K79 efficiently blocked the replication of HBV, and interference with GCN5 resulted in decreased levels of HBV DNA, HBsAg, and HBeAg in the supernatant from de novo HBV-infected HepaRG cells. Consistently, the levels of histone H3K79 succinylation were significantly elevated in the livers of HBV-infected human liver-chimeric mice. The knockdown or overexpression of GCN5 or the mutant of GCN5 could affect the binding of GCN5 to cccDNA or H3K79 succinylation, leading to a change in cccDNA transcription activity. In addition, Southern blot analysis validated that siGCN5 decreased the levels of cccDNA in the cells, suggesting that GCN5-mediated succinylation of histone H3K79 contributes to the epigenetic regulation of cccDNA minichromosome. Strikingly, IFN-α effectively depressed histone H3K79 succinylation in HBV cccDNA minichromosome in de novo HepG2-NTCP and HBV-infected HepaRG cells.ConclusionsIFN-α epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA. Our findings provide new insights into the mechanism by which IFN-α modulate the epigenetic regulation of HBV cccDNA minichromosome.

Dataset Information

Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.

Publications

Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.

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