Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors
5 affiliations
- 1Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, United States
- 2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, United States
- 3Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15219, United States
- 4Cancer Therapeutics Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, United States
- 5Department of Computational & Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15219, 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.
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BibTeX
@article{zhang2026development,
title = {{Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors}},
author = {Hu Zhang and Laura L. Vollmer and Ning Sun and Cheng-Der Liu and Mark E. Schurdak and Andreas Vogt and Haitao Guo},
journal = {Antiviral research},
year = {2026},
month = aug,
doi = {10.1016/j.antiviral.2026.106508},
url = {https://doi.org/10.1016/j.antiviral.2026.106508},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors
AU - Hu Zhang
AU - Laura L. Vollmer
AU - Ning Sun
AU - Cheng-Der Liu
AU - Mark E. Schurdak
AU - Andreas Vogt
AU - Haitao Guo
PY - 2026
DA - 2026/08/07
JO - Antiviral research
DO - 10.1016/j.antiviral.2026.106508
UR - https://doi.org/10.1016/j.antiviral.2026.106508
AB - 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.
SN - 0166-3542
SN - 1872-9096
ER -