Dual HBV cccDNA reporter hepatocyte models enable high-throughput identification of cccDNA modulators

Chronic hepatitis B remains difficult to cure because the viral covalently closed circular DNA (cccDNA) minichromosome can persist and sustain viral transcription, creating a need for scalable, quantitative cccDNA-linked readouts for high-throughput screening (HTS) of cccDNA-modulating agents. Here, we developed two complementary hepatocyte reporter models that couple a split-NanoLuc HiBiT signal to cccDNA (or recombinant cccDNA, rcccDNA) activity: a replication-competent HBV reporter in HepaRG cells (HepaRG-Hibit16) generated by inserting HiBiT tags into the precore region to produce a secreted luminescent readout aligned with cccDNA-driven expression, and a Cre/Lox-based rcccDNA reporter in HepG2 cells (HepG2-Rccc1a) that rapidly generates rcccDNA episomes with a matched HiBiT readout. Screening 1,403 FDA-approved compounds across both models identified 13 concordant, non-cytotoxic hits. As an exemplar concordant hit, palovarotene (a retinoic acid receptor-γ agonist) reduced HBV antigens, HBV DNA, and cccDNA and inhibited HBV infection in multiple hepatocyte-based in vitro systems. Together, this dual-reporter strategy enables efficient HTS and cross-validation to prioritise candidate cccDNA transcription modulators and streamline early-stage HBV discovery workflows focused on cccDNA-directed interventions.