title: Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells.
abstract: A direct involvement of the hepatitis B virus (HBV) preS1-(21-47) sequence in virus attachment to cell membrane receptor(s) and the presence on the plasma membranes of HepG2 cells of protein(s) with receptor activity for HBV have been suggested by many previous experiments. In this study, by using a tetravalent derivative of the preS1-(21-47) sequence, we have isolated by affinity chromatography from detergent-solubilized HepG2 plasma membranes a 44-kDa protein (HBV-binding protein; HBV-BP), which was found to closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors. Comparison of SCCA1 sequence with the sequence of the corresponding HBV-BP cDNA, cloned by polymerase chain reaction starting from RNA poly(A)(+) fractions extracted from HepG2 cells, indicated the presence of only four nucleotide substitutions in the coding region, leading to three amino acid changes. Intact recombinant HBV-BP lacked inhibitory activity for serine proteases such as alpha-chymotrypsin and trypsin but inhibited with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP after transfection of corresponding cDNA showed a virus binding capacity increased by 2 orders of magnitude compared with untransfected cells, while Chinese hamster ovary cells, which normally do not bind to HBV, acquired susceptibility to HBV binding after transfection. Native HBV particle entry was enhanced in transfected cells. Both recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and internalization in transfected cells as well as in primary human hepatocytes in a dose-dependent manner. Our findings suggest that this protein plays a major role in HBV infection.
authors.affiliation: TECNOGEN S.C.p.A., Parco Scientifico, 81015 Piana di Monte Verna (CE), Caserta 81015, Italy.
authors.forename: S
authors.initials: S
authors.lastname: De Falco
authors.forename: M G
authors.initials: MG
authors.lastname: Ruvoletto
authors.forename: A
authors.initials: A
authors.lastname: Verdoliva
authors.forename: M
authors.initials: M
authors.lastname: Ruvo
authors.forename: A
authors.initials: A
authors.lastname: Raucci
authors.forename: M
authors.initials: M
authors.lastname: Marino
authors.forename: S
authors.initials: S
authors.lastname: Senatore
authors.forename: G
authors.initials: G
authors.lastname: Cassani
authors.forename: A
authors.initials: A
authors.lastname: Alberti
authors.forename: P
authors.initials: P
authors.lastname: Pontisso
authors.forename: G
authors.initials: G
authors.lastname: Fassina
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D002845:Chromatography; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006515:Hepatitis B virus / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D011061:Poly A / Q000378:metabolism; D011480:Protease Inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011991:Receptors, Virus / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015843:Serpins; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D011480:Protease Inhibitors; D011991:Receptors, Virus; D011994:Recombinant Proteins; D015843:Serpins; C438016:hepatitis B virus-binding protein, human; C051072:squamous cell carcinoma-related antigen; D011061:Poly A; D002403:Cathepsins; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; D010206:Papain