Characterization of Intracellular Precore-Derived Proteins and Their Functions in Hepatitis B Virus-Infected Human Hepatocytes

Hepatitis B virus (HBV) precore protein is not essential for viral replication but is thought to facilitate chronic infection. In addition to the secreted precore products, including the hepatitis B e antigen (HBeAg) and PreC protein, intracellular precore-derived proteins in HBV-infected human hepatocytes remain poorly characterized, and their roles, if any, remain largely unknown. Here, we detected multiple precore derivatives, including the nonprocessed precursor p25 and the processing intermediate p22, in HBV-infected human hepatocytes as well as human hepatoma cells overexpressing the HBV precore protein. Both p25 and p22 showed phosphorylated and unphosphorylated forms, which were located in different intracellular compartments. Interestingly, precore expression was associated with decreases in intracellular HBV core protein (HBc) and secreted DNA-containing virions but was also associated with an increase in secreted empty virions. The decrease in HBc by precore could be attributed to cytosolic p22, which caused HBc degradation, at least in part by the proteasome, and consequently decreased HBV pregenomic RNA packaging and DNA synthesis. In addition, cytosolic p22 formed chimeric capsids with HBc in the cell, which were further secreted in virions. In contrast, the PreC antigen, like HBeAg, was secreted via the endoplasmic reticulum (ER)-Golgi secretory pathway and was thus unable to form capsids in the cell or be secreted in virions. Furthermore, p25, as well as p22, were secreted in virions from HBV-infected human hepatocytes and were detected in the sera of HBV-infected chimpanzees. In summary, we have detected multiple intracellular precore-derived proteins in HBV-infected human hepatocytes and revealed novel precore functions in the viral life cycle.