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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.
Researchers developed a new cell line, HepBLE12, to detect Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). This breakthrough enables high-throughput screening for new antiviral therapies targeting persistent HBV infections.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- Hepatitis B virus (HBV) persistence is primarily maintained by the cccDNA minichromosome, a significant obstacle for curative antiviral treatments.
- Current limitations in detecting cccDNA and identifying surrogate markers impede the development of targeted antiviral therapies and high-throughput screening (HTS).
Purpose of the Study:
- To establish a novel, inducible, cccDNA-dependent nanoluciferase (NLuc) reporter cell line for HTS.
- To develop a reliable method for directly quantifying cccDNA activity for antiviral drug discovery.
Main Methods:
- Engineered HepBLE12 cells by inserting a split-NLuc HiBiT tag into the HBV precore region of a transgene.
- Utilized the HiBiT assay for direct detection of intracellular HiBiT-pC protein, correlating luminescence with cccDNA activity.
- Optimized and miniaturized the assay to a 384-well format for HTS, including automation and validation studies.
Main Results:
- The HepBLE12 cell line demonstrated inducible and robust cccDNA-dependent reporter activity.
- The HiBiT assay provided a direct measure of cccDNA activity, distinct from secreted markers like HBeAg.
- The HTS platform achieved excellent performance metrics (S/B ≈ 9, CV < 10%, Z-factor = 0.74), meeting HTS quality benchmarks.
Conclusions:
- The developed HepBLE12 cell-based HTS platform offers a robust and practical tool for identifying novel inhibitors of HBV cccDNA.
- This system facilitates the discovery of antiviral therapies aimed at eliminating persistent HBV infections.
