Primary cultured normal human hepatocytes for hepatitis B virus receptor studies.
OBJECTIVE
We analyzed the hepatitis B virus envelope specificities (HBs, preS2 and preS1) involved in virus attachment to normal human hepatocytes, and we performed in vitro hepatitis B virus infection experiments without addition of dimethyl sulfoxide and polyethylene glycol, which may affect cell membrane integrity, in order to study further the early steps of the life cycle of the hepatitis B virus.


METHODS
Primary normal human hepatocytes were prepared from surgical biopsies by the two-step collagenase perfusion technique, and cultured in a fetal calf serum-free medium supplemented with 10(-6) M dexamethasone. Cell-binding assays, ligand blotting and immunohistochemistry experiments were carried out using our anti-idiotypic (Ab2) antibodies (Ab2s/preS1, Ab2s/preS2 and Ab2s/HBs).


RESULTS
Probing primary normal human hepatocytes, the 35-kDa major preS1-binding protein (preS1-BP35) we have previously identified in human hepatoma HepG2 cells was recognized in blotting, whereas both HBs- and preS1-specificities of the hepatitis B virus envelope interacted strongly with normal human hepatocyte cell membrane in cell-binding assays and immunohistochemistry experiments. Hepatitis B virus infectivity studies confirmed a great inter-experimental variability depending on donors and liver perfusion, and demonstrated a great intra-experimental variability depending on the serum-derived hepatitis B virus isolate used for the inoculation. In our culture conditions, only increased detection of the RC and CCC DNA forms of hepatitis B virus in cells and of hepatitis B virus surface antigens in medium was observed 4 to 8 days after exposure of cells to hepatitis B virus.


CONCLUSIONS
These findings support a potential role for preS1-BP35 as a receptor protein for hepatitis B virus. In our hands, limitation(s) in the hepatitis B virus life cycle may occur at some step after virion binding, and likely result from complex regulation of reverse transcription of the RNA and translation of core protein by extrahepatic host factors or/and by the virus itself. However, the normal human hepatocyte model developed here is available for studying the initial steps in hepatitis B virus entry into cells.
Mabit
H
H
Institut National de la Santé de la Recherche Médicale Unité, Unité, Clamart, France.
Vons
C
C
Dubanchet
S
S
Capel
F
F
Franco
D
D
Petit
M A
MA
D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis; D006509:Hepatitis B / Q000276:immunology / Q000378:metabolism; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D011498:Protein Precursors / Q000032:analysis; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012016:Reference Values; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen