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Journal article
Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators
Antiviral research, v 132
01 Aug 2016
PMID: 27185623
Featured in Collection : UN Sustainable Development Goals @ Drexel
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. (C) 2016 Elsevier B.V. All rights reserved.
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Details
- Title
- Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators
- Creators
- Dawei Cai - Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA, 18902, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.Xiaohe Wang - Baruch S. Blumberg InstituteRan Yan - Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA, 18902, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.Richeng Mao - Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, PA, 18902, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.Yuanjie Liu - Indiana UniversityChanghua Ji - Virology Discovery and Translational Area, Roche Pharma Research and Early Development, Nutley, NJ, 07110, USA.Andrea Cuconati - Baruch S. Blumberg InstituteHaitao Guo - Indiana University
- Publication Details
- Antiviral research, v 132
- Publisher
- Elsevier
- Number of pages
- 12
- Grant note
- RPF-200 / Roche; Roche Holding R01AI094474; R01AI110762 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA R01AI110762 / NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Allergy & Infectious Diseases (NIAID)
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Microbiology and Immunology
- Web of Science ID
- WOS:000382345100004
- Scopus ID
- 2-s2.0-84971233976
- Other Identifier
- 991019168642704721
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- Collaboration types
- Industry collaboration
- Domestic collaboration
- Web of Science research areas
- Pharmacology & Pharmacy
- Virology