Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.