Development of a cellular high-content, immunofluorescent HBV core assay to identify novel capsid assembly modulators that induce the formation of aberrant HBV core structures

•High-content HBV core assay to identify novel capsid assembly modulators.•High throughput screen using a combined biochemical and cellular approach.•Aggregated core structures detected by using image analysis. Hepatitis B Virus (HBV) core protein has multiple functions in the viral life cycle and is an attractive target for new anti-viral therapies. Capsid assembly modulators (CAMs) target the core protein and induce the formation of either morphologically normal (CAM-N) or aberrant structures (CAM-A), both devoid of genomic material. To date a diverse family of CAM-N chemotypes has been identified, but in contrast, described CAM-As are based on the heteroaryldihydropyrimidine (HAP) scaffold. We used the HBV-inducible HepG2.117 cell line with immunofluorescent labeling of HBV core to develop and validate a cellular high-content image-based assay where aggregated core structures are identified using image analysis spot texture features. Treatment with HAPs led to a dose- and time-dependent formation of aggregated core appearing as dot-like structures in the cytoplasm and nucleus. By combining a biochemical and cellular screening approach, a compound was identified as a novel non-HAP scaffold able to induce dose-dependent formation of aberrant core structures, which was confirmed by electron microscopy and native gel electrophoresis. This compound displayed anti-HBV activity in HepG2.117 cells, providing proof-of-concept for our screening approach. We believe our combined biochemical and cellular high-content screening method will aid in expanding the range of CAM-A chemotypes.