Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells

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 pl asma 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 c orrespond to the human squamous cell carcinoma antigen I (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 poly merase 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 recombina nt HBV-BP lacked inhibitory activity for serine proteases such as alpha -ch ymotrypsin and trypsin but inhibited with high potency cysteine proteases s uch as papain and cathepsin L. Direct binding experiments confirmed the int eraction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overe xpressing HBV-BP after transfection of corresponding cDNA showed a virus bi nding capacity increased by 2 orders of magnitude compared with untransfect ed 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 a nd antibodies to recombinant HBV-BP blocked virus binding and internalizati on in transfected cells as well as in primary human helpatocytes in a dose- dependent manner. Our findings suggest that this protein plays a major role in HBV infection.