Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein p recursor by host signal peptidase cleavages, These glycoproteins interact t o form a noncovalent heterodimeric complex, which is retained in the endopl asmic reticulum, HCV glycoproteins, E1 and E2, are heavily modified by N-li nked glycosylation, A recent study has revealed that upon partial deglycosy lation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized, In this work, the unused glycos ylation site on the E1 glycoprotein was identified and the influence of N-l inked glycosylation on the formation of the HCV glycoprotein complex was st udied by expressing a panel of E1 glycosylation mutants in HepG2 cells, Eac h of the five potential N-linked 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, Ex pression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharid es and the Pro immediately following the sequon (Asn-Trp-Ser) precludes cor e glycosylation, The effect of each mutation on the formation of noncovalen t E1E2 complexes was also analysed, As determined with the use of a conform ation-sensitive monoclonal antibody, mutations at positions N2 and N3 had n o, or only minor, effects on the assembly of the E1E2 complex, whereas a mu tation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.