Hepatitis C virus glycoprotein E2 binding to CD81: The role of E1E2 cleavage and protein glycosylation in bioactivity

The hepatitis C Virus glycoproteins E1 and 2 have been expressed using reco mbinant baculoviruses following fusion to the carrier protein glutathione S -transferase (GST). Proteins were expressed singly and as an E1 E2 polyprot ein with and without an N-terminal affinity tag. Expression of the E1E2 pol yprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutath ione affinity chromatography. Baculovirus expressed E2 and a purified GST-E 1E2 protein bound to the second extracellular loop of CD81 (EC2), a reporte d ligand for the molecule. but not to a truncated derivative of CD81 consis ting of only the central domain of the loop. Purified GST-E2, however, fail ed to bind to CD81 suggesting a requirement for a free E2 amino terminus fo r biological activity. The binding to CD81 by baculovirus expressed E2 prot ein was comparable to that observed for E2 derived from mammalian cells whe n detected by a monoclonal antibody sensitive to protein conformation. Furt hermore, E2 protein expressed in insect cells in the presence of N-butyldeo xynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with El and bound to CD81-EC2 similarly to untreated protein. Tog ether these data suggest that although hyperglucosylation of E2 does not ha ve a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required. (C) 2000 Academic Press.