title: 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.
authors.affiliation: 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.
authors.forename: Dawei
authors.initials: D
authors.lastname: Cai
authors.affiliation: Baruch S. Blumberg Institute, Hepatitis B Foundation, Doylestown, PA, 18902, USA.
authors.forename: Xiaohe
authors.initials: X
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Ran
authors.initials: R
authors.lastname: Yan
authors.affiliation: 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.
authors.forename: Richeng
authors.initials: R
authors.lastname: Mao
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Virology Discovery and Translational Area, Roche Pharma Research and Early Development, Nutley, NJ, 07110, USA. Electronic address: changhua.ji2@pfizer.com.
authors.forename: Changhua
authors.initials: C
authors.lastname: Ji
authors.affiliation: Baruch S. Blumberg Institute, Hepatitis B Foundation, Doylestown, PA, 18902, USA. Electronic address: acuconati@arbutusbio.com.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cuconati
authors.affiliation: 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. Electronic address: haitguo@iupui.edu.
authors.forename: Haitao
authors.initials: H
authors.lastname: Guo
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D004270:DNA, Circular; D004279:DNA, Viral; D055808:Drug Discovery; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016679:Genome, Viral; D006513:Hepatitis B e Antigens / Q000235:genetics* / Q000276:immunology*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000276:immunology*; D057166:High-Throughput Screening Assays; D006801:Humans; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins; D014158:Transcription, Genetic; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D004270:DNA, Circular; D004279:DNA, Viral; D000939:Epitopes; D006513:Hepatitis B e Antigens; D011993:Recombinant Fusion Proteins
keywords: HBeAg; Hepatitis B virus; High throughput screening; cccDNA