A hepatitis B virus-free cccDNA-producing stable cell for antiviral screening

The covalently closed circular DNA (cccDNA) of the Hepatitis B virus (HBV) serves as a template for producing progeny viruses in virally infected hepatocytes. Promising cccDNA-targeting antiviral agents remain unavailable and unpredictable in the research and development pipelines, making sterile HBV elimination challenging at the current stage. The major challenge of discriminating trace amounts of cccDNA from the abundant HBV relaxed circular DNA (rcDNA), which is nearly identical to cccDNA in sequence, substantially discourages efforts to discover and directly screen cccDNA-targeting drugs. Therefore, an easy cccDNA cell culture system is required for high-throughput drug screening. In this study, we designed an HBV cccDNA self-generating stable cell culture system using a functional complementary concept and successfully generated an HBV cccDNA Gaussia luciferase reporter cell line in HepG2 and Huh7 cells. This design ensures that the Gluc signal is exclusively expressed upon cccDNA formation, allowing for the accurate and easy measurement of cccDNA levels via luminescent signals. Using this system, in conjunction with a firefly luciferase reporter to monitor cell activity, we screened 2074 drugs in the HepG2-HBV-cccDNA/Firefly cell line. Four compounds were selected for further experimentation and their anti-HBV effects were confirmed. Thus, this virus-free hepatitis B cccDNA cell culture system provides a valuable and convenient platform for the high-throughput screening of anti-HBV drugs.