PROCESSING OF THE E1 GLYCOPROTEIN OF HEPATITIS-C VIRUS EXPRESSED IN MAMMALIAN-CELLS

The structural part of the hepatitis C virus (HCV) genome encodes a ca psid protein, C, and two envelope glycoproteins, E1 and E2, released f rom the virus polyprotein precursor by signalase(s) cleavage(s). The p rocessing of E1 was investigated by infecting simian cells with recomb inant vaccinia viruses expressing parts of the HCV structural proteins . When the predicted E1 sequence was expressed alone (amino acid resid ues 174-370 of the polyprotein) or with the capsid protein gene (resid ues 1-370), it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a tru ncated C-E1-truncated E2 polypeptide (residues 132-383), the processed E1 product had the expected apparent molecular mass of 31 kDa, sugges ting that flanking sequences are necessary for the generation of the m ature 31 kDa E1 form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed tha t, in both species, only four of the five potential N-linked glycosyla tion sites were recognized, indicating that glycosylation was not invo lved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371-3 83, defined as the E2 signal sequence, may be responsible for the diff erence in electrophoretic mobility of the two E1 species. In vitro tra nslation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.