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Updated: Aug 9, 2026
Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle
Published on: February 1, 2017
Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors
Hu Zhang1, Laura L Vollmer2, Ning Sun1
1Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, United States; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, United States.
Abstract:
Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12 cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B) ≈ 9, coefficient of variance (CV) < 10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.
Insights
Researchers developed a new cell line, HepBLE12, for detecting Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). This breakthrough enables high-throughput screening (HTS) for new antiviral therapies targeting persistent HBV infections.
Area of Science:
- Hepatology
- Virology
- Molecular Biology
Background:
- Hepatitis B virus (HBV) persistence is primarily maintained by the stable HBV covalently closed circular DNA (cccDNA) minichromosome.
- The lack of sensitive methods for detecting cccDNA and reliable surrogate markers impedes the development of curative antiviral strategies.
- Targeting cccDNA is crucial for eliminating persistent HBV infection, but requires effective screening tools.
Purpose of the Study:
- To establish a novel, inducible, and cccDNA-dependent reporter cell line for high-throughput screening (HTS).
- To develop a quantitative assay for directly measuring cccDNA activity and facilitate drug discovery.
- To create a robust platform for identifying inhibitors that target the HBV cccDNA minichromosome.
Main Methods:
- Developed the HepBLE12 cell line by inserting a split-nanoluciferase (NLuc) HiBiT tag into the precore region of an HBV transgene.
- Utilized the HiBiT tag for direct detection of intracellular viral protein, correlating with cccDNA transcriptional activity.
- Miniaturized and optimized the assay for a 384-well format, enabling automation and HTS.
- Validated the assay's performance through variability studies and a pilot screen, assessing signal-to-background, coefficient of variance, and Z-factor.
Main Results:
- The HepBLE12 cell line demonstrated inducible and robust HBV replication dependent on cccDNA.
- The HiBiT assay provided a direct measure of cccDNA activity, distinct from secreted markers like HBeAg.
- The optimized HTS assay exhibited excellent performance metrics: S/B ≈ 9, CV < 10%, and Z-factor = 0.74.
- The platform proved suitable for automated screening and demonstrated reliability in multi-day studies.
Conclusions:
- The established HepBLE12 cell-based HTS platform is a robust and practical tool for identifying novel inhibitors of HBV cccDNA.
- This reporter system overcomes limitations of previous methods, offering a direct and quantitative measure of cccDNA activity.
- The developed platform significantly advances the search for curative therapies against persistent Hepatitis B virus infections.
