Development of a CRE/CREB-driven HBx responsive HBV cell culture reporter system for antiviral drug evaluation.

Background & Aims: Chronic HBV infection is a leading cause of liver disease and cancer. Current therapies fail to eliminate covalently closed circular DNA (cccDNA), underscoring the need for novel strategies. We aimed to develop a quantitative cell-based reporter system that detects HBx expression during HBV infection and is suitable for screening compounds with anti-HBV activity. Methods: We generated an HBx-responsive cell line stably expressing nano-luciferase (nLuc) under the control of a viral cAMP-response element (vCRE) derived from the human T-cell leukemia virus-1 core promoter. The system was used to evaluate various drug inhibitors in HBV-infected cells. Results: The vCRE-nLuc system was confirmed to be responsive to HBx using pHBV1.3-wild-type, pHBV1.3-null-X and pMyc-HBx constructs (p <0.0001). HBV infection with both culture-derived and patient-derived clinical isolates (genotypes A-E) significantly increased luciferase activity (p <0.0001). Treatment with HBx inhibitors (siRNA, specific HBx inhibitors), as well as IFNα reduced luciferase production (p <0.0001). In contrast, antivirals that do not interfere with protein production (tenofovir), or viral entry (bulevirtide) showed no effect (p <0.05), underscoring the specificity of the system for identifying compounds that inhibit protein production and/or HBx function. Finally, HepG2.2.15 cells carrying HBV and transduced with vCRE-nLuc confirmed the system's suitability for monitoring HBV infection. Conclusions: We established a vCRE-nLuc-driven HBx-responsive cell line for quantitative monitoring of HBV infection and evaluation of antiviral drugs. This system holds potential for identifying new anti-HBV agents and advancing our understanding of HBV replication. Impact and implications: The HBx-responsive vCRE-nLuc reporter system provides a sensitive and scalable platform to monitor HBV infection and evaluate antiviral compounds targeting HBx function. Its ability to detect HBx activity from both laboratory and clinical HBV isolates underscore its translational relevance. By enabling selective screening of HBx-targeting agents, this system may advance efforts to silence covalently closed circular DNA and accelerate the development of curative therapies for chronic hepatitis B.