Hepatitis C virus (HCV) glycoproteins (E1 and E2)both contain a carboxy-terminal hydrophobic region, which presumably serves as a membrane anchor. When they are expressed in animal cell cultures, these glycoproteins, in both mature complexes and misfolded aggregates, are retained in the endoplasmic reticulum. The effect of carboxyterminal deletions on HCV glycoprotein secretion and folding was examined in this study. Sindbis and/or vaccinia virus recombinants expressing truncated forms of these glycoproteins ending at amino acids 311, 330, 354 and 360 (truncated E1), and 661, 688, 704 and 715 (truncated E2) were constructed. When expressed using Sindbis virus vectors, only truncated forms of E1 and E2 ending at amino acids 311 (E1 t311 ) and 661 (E2 t661 ), respectively, were efficiently secreted. Analysis of

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by Nlinked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential Nlinked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.