PRIMARY CULTURED NORMAL HUMAN HEPATOCYTES FOR HEPATITIS-B VIRUS RECEPTOR STUDIES

Background/Aims: We analyzed the hepatitis B virus envelope specificit ies (HBs, preS2 and preS1) involved in virus attachment to normal huma n hepatocytes, and we performed in vitro hepatitis B virus infection e xperiments without addition of dimethyl sulfoxide and polyethylene gly col, which may affect cell membrane integrity, in order to study furth er the early steps of the life cycle of the hepatitis B virus. Methods : Primary normal human hepatocytes were prepared from surgical biopsie s by the two-step collagenase perfusion technique, and cultured in a f etal calf serum-free medium supplemented with 10(-6) M dexamethasone. Cell-binding assays, ligand blotting and immunohistochemistry experime nts were carried out using our anti-idiotypic (Ab2) antibodies (Ab2s/p reS1, Ab2s/preS2 and Ab2s/HBs). Results: Probing primary normal human hepatocytes, the 35-kDa major preS1-binding protein (preS1-BP35) we ha ve previously identified in human hepatoma HepG2 cells was recognized in blotting, whereas both HBs- and preS1-specificities of the hepatiti s B virus envelope interacted strongly with normal human hepatocyte ce ll membrane in cell-binding assays and immunohistochemistry experiment s. Hepatitis B virus infectivity studies confirmed a great inter-exper imental variability depending on donors and liver perfusion, and demon strated a great intra-experimental variability depending on the serum- derived hepatitis B virus isolate used for the inoculation. In our cul ture conditions, only increased detection of the RC and CCC DNA forms of hepatitis B virus in cells and of hepatitis B virus surface antigen s in medium was observed 4 to 8 days after exposure of cells to hepati tis B virus. Conclusion: These findings support a potential role for p reS1-BP35 as a receptor protein for hepatitis B virus. In our hands, l imitation(s) in the hepatitis B virus life cycle may occur at some ste p after virion binding, and likely result from complex regulation of r everse transcription of the RNA and translation of core protein by ext rahepatic host factors or/and by the virus itself. However, the normal human hepatocyte model developed here is available for studying the i nitial steps in hepatitis B virus entry into cells.