Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes

Superior cell culture models for hepatitis B virus (HBV) will help advance insights into host-virus interactions. To identify mechanisms regulating HBV replication, this study used cultured human HepG2 cells and adult or fetal hepatocytes transduced with adenoviral vector to express HBV upstream of green fluorescent protein. The vector efficiently transduced all cell types. In HepG2 cells, replicative viral intermediates, nucleocapsid-associated HBcAg, and HBsAg were expressed. However, in fetal or adult hepatocytes, pregenomic HBV RNA and viral RNAs were expressed, but nucleocapsid-associated HBcAg in cells or HBsAg in culture medium were absent, indicating interruptions in viral replication due to possible microRNA-related interference. MicroRNA profiling demonstrated that a large number of microRNAs with antiviral potential were differentially expressed in hepatocytes after culture. In transfection assays using HepG2 cells, candidate antiviral microRNAs, e.g., hsa-miR-24 or hsa-miR-638 decreased the levels of HBV transcripts or HBV gene products. Since candidate microRNAs could have targeted interferon response genes as an alternative explanation interferon signaling was examined. However, HBV replication in cultured hepatocytes was not restored despite successful inhibition of JAK1/2-STAT signaling by the inhibitor, ruxolitinib. Therefore, HBV was unable to complete replication in cultured hepatocytes due to expression of multiple antiviral microRNAs. This mechanism should help understand restrictions in HBV replication for developing HBV models in cultured cells while providing frameworks for pathophysiological studies of HBV replication in subsets of hepatocytes or stem/progenitor cells during hepatitis.