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 membranereceptor(s) and the presence on the plasma membranesof HepG2 cells of protein(s) with receptor activityfor HBV have been suggested by many previousexperiments. In this study, by using a tetravalent derivativeof the preS1-(21-47) sequence, we have isolated byaffinity chromatography from detergent-solubilizedHepG2 plasma membranes a 44-kDa protein (HBV-bindingprotein; HBV-BP), which was found to closely correspondto the human squamous cell carcinoma antigen 1(SCCA1), a member of the ovalbumin family of serineprotease inhibitors. Comparison of SCCA1 sequencewith the sequence of the corresponding HBV-BP cDNA,cloned by polymerase chain reaction starting from RNApoly(A) fractions extracted from HepG2 cells, indicatedthe presence of only four nucleotide substitutions in thecoding region, leading to three amino acid changes. Intactrecombinant HBV-BP lacked inhibitory activity forserine proteases such as -chymotrypsin and trypsinbut inhibited with high potency cysteine proteases suchas papain and cathepsin L. Direct binding experimentsconfirmed the interaction of recombinant HBV-BP withthe HBV preS1 domain. HepG2 cells overexpressingHBV-BP after transfection of corresponding cDNAshowed a virus binding capacity increased by 2 orders ofmagnitude compared with untransfected cells, whileChinese hamster ovary cells, which normally do notbind to HBV, acquired susceptibility to HBV bindingafter transfection. Native HBV particle entry was enhancedin transfected cells. Both recombinant HBV-BPand antibodies to recombinant HBV-BP blocked virusbinding and internalization in transfected cells as wellas in primary human hepatocytes in a dose-dependentmanner. Our findings suggest that this protein plays amajor role in HBV infection.