In vitro resistance to interferon-alpha of hepatitis B virus with basic core promoter double mutation

The hepatitis B virus (HBV) genome basic core promoter (BCP) modulates HBeAg secretion at the transcriptional level. In addition to pre-core mutations, variations in the BCP are related to hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. HBeAg-negative chronic hepatitis B patients show a lower sustained response to interferon (IFN). The aim of this study was to determine if there is a relationship between HBV BCP mutation and sensitivity of HBV to IFN-alpha in vitro. BCP mutations were introduced by site-directed mutagenesis and the entire genomes of wild-type and mutant HBV were transiently transferred into Huh7 cells by calcium phosphate transfection. With or without IFN-alpha, viral products in the culture medium and viral replication intermediates in the cytoplasm were detected 3 days after transfection. The amount of hepatitis B surface antigen (HBsAg) secreted by wild-type HBV and the BCP mutant was similar, while HBeAg secreted by the mutant was decreased by 35.4%. HBV particles and replication intermediates of the BCP mutant were increased. After IFN-alpha was added, HBeAg, HBV DNA and HBV replication intermediates decreased for both the wild-type HBV (by 25.7%, 31.8%, 29.8%, respectively) and the BCP mutant (by 8.4%, 27.4%, 10.1%, respectively). These data indicate that HBV harboring the BCP double mutation has stronger replication competence and lower sensitivity to IFN-alpha than wild-type.