Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins

Abstract

Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.

FIG. 1.

Analysis of E1 glycosylation mutants and their effects on the formation of E1E2 complexes. (A) Schematic representation of N-glycosylation sites in HCV glycoprotein E1. The positions of the glycosylation sites are indicated above the box corresponding to the E1 protein. The numbers correspond to the positions in the polyprotein of reference strain H (GenBank accession no. AF009606). The mutants are named with an N followed by a number related to the position of the glycosylation site on the map. The transmembrane domain of E1 is indicated with a dark box. (B and C) Role of E1 glycans in the formation of E1E2 complexes. A plasmids encoding a wild-type (WT) or mutant E1 protein in the context of an E1E2 polyprotein was transfected into 293T cells. A control experiment was performed with 293T cells transfected with a plasmid encoding the feline endogenous retrovirus RD114 glycoprotein (Ctrl) (46). At 24 h posttransfection, the cells were pulse labeled with [³⁵S]methionine-[³⁵S]cysteine for 30 min and then chased for 4 h. Cell lysates were used for immunoprecipitation with the MAbs A4 (anti-E1) (B) and H53 (anti-E2) (C). Immunoprecipitates were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. E1-3g corresponds to E1 lacking one glycan. The HCV envelope proteins E1 and E2 as well as SDS-resistant E1E2 complexes are indicated on the right. (D) Percentages of noncovalent E1E2 heterodimers. The intensities of the bands corresponding to the E2 proteins precipitated by MAbs H53 and A4 were measured by phosphorimaging for three independent experiments, and the mean percentage of noncovalent E1E2 heterodimers was cal-culated for each mutant as follows: (amount of E2 protein from the mutant precipitated by MAb H53/amount of E2 from the mutant precipitated by MAb A4)/(amount of wild-type E2 precipitated by MAb H53/amount of wild-type E2 precipitated by MAb A4).

FIG. 2.

Analysis of E2 glycosylation mutants and their effects on the formation of E1E2 complexes. (A) Schematic representation of N-glycosylation sites in HCV glycoprotein E2. The positions of the glycosylation sites are indicated above the box corresponding to the E2 protein. The numbers correspond to the positions in the polyprotein of reference strain H (accession no. AF009606). The mutants are named with an N followed by a number related to the position of the glycosylation site on the map. The transmembrane domain of E2 is indicated with a dark box. (B) Analysis of N-linked glycosylation mutants of E2. Plasmids containing the sequences encoding mutants E2N1 to E2N11 were transiently expressed in 293T cells. At 16 h posttransfection, cell lysates were separated by SDS-PAGE (10% polyacrylamide) and the proteins of interest were revealed by Western blotting with an anti-E2 (3/11) MAb. A cell lysate containing the wild-type E1 and E2 proteins (WT) was run in parallel. E2-10g corresponds to E2 lacking one glycan. (C and D) Role of E2 glycans in the formation of E1E2 complexes. A plasmid encoding a wild-type (WT) or mutant E2 protein in the context of an E1E2 polyprotein was transfected into 293T cells. A control experiment was performed with 293T cells transfected with a plasmid encoding the RD114 retroviral envelope protein (Ctrl) (46). At 24 h posttransfection, the cells were pulse labeled with [³⁵S]methionine-[³⁵S]cysteine for 30 min and then chased for 4 h. Cell lysates were used for immunoprecipitation with the MAbs 3/11 (C) and H53 (D), and the immunoprecipitates were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. HCV envelope proteins E1 and E2, as well as SDS-resistant E1E2 complexes, are indicated on the right. (E) Percentages of noncovalent E1E2 heterodimers. The intensities of the bands corresponding to the E2 proteins precipitated by MAbs H53 and 3/11 were measured by phosphorimaging for three independent experiments, and the mean percentage of noncovalent E1E2 heterodimers was calculated for each mutant as follows: (amount of mutant E2 protein precipitated by MAb H53/amount of mutant E2 precipitated by MAb 3/11)/(amount of wild-type E2 precipitated by MAb H53/amount of wild-type E2 precipitated by MAb 3/11).

FIG. 3.

Interaction of HCV glycosylation mutants with human CD81LEL. A plasmid encoding a wild-type (WT) or mutant E2 protein in the context of an E1E2 polyprotein was transfected into 293T cells. At 24 h posttransfection, the cells were lysed and analyzed in a CD81LEL-GST pull-down assay (A) using human (h) or murine (m) CD81LEL fused to GST. The presence of the E2 glycoprotein in the precipitates was revealed by Western blotting with an anti-E2 MAb (3/11). The expression of mutant proteins was verified by direct Western blotting of cell lysates (B).

FIG. 4.

Role of E1 glycans in HCVpp infectivity. (A) Plasmids encoding E1 mutants in the context of an E1E2 polyprotein were used to generate HCVpp (E1N1 to E1N4). Control experiments in the absence of HCV envelope proteins were also performed (−env). Infection assays with the luciferase reporter gene were performed by using target Huh-7 human hepatocarcinoma cells. Similar inputs of viral particles were used in each experiment, and this was confirmed by comparing the amounts of capsid protein incorporated into HCVpp (see panel B, anti-Gag). The results are expressed as percentages of infectivity. For each mutant, the percentage of infectivity was calculated as the luciferase activity of HCVpp produced with mutant envelope proteins divided by the luciferase activity of HCVpp produced with wild-type E1 and E2 (WT). The results are reported as means ± standard deviations of six independent experiments. Pseudotyped particles produced in the absence of envelope proteins were used as a control. The mean luciferase activity of such pseudotyped particles produced in the absence of envelope proteins represented <2% of the activity measured for HCVpp. The luciferase activities (in relative light units) in a representative experiment were as follows: WT, 3.6 × 10⁵; N1, 1.5 × 10⁵; N2, 3.4 × 10⁴; N3, 2.7 × 10⁵; and N4, 7.7 × 10⁴. (B) Incorporation of HCV envelope proteins into HCVpp. Particles were pelleted through 30% sucrose cushions and analyzed by Western blotting. HCV envelope glycoproteins and the capsid protein of MLV were revealed with the following specific MAbs: anti-E1 (A4), anti-E2 (3/11), and anti-MLV capsid (R187, anti-Gag). The expression of mutant proteins was verified by direct Western blotting of cell lysates. The proteins analyzed for this figure were separated by 15% SDS-PAGE, and for this reason, the differences in size between the mutant and wild-type proteins were reduced.

FIG. 5.

Role of E2 glycans in HCVpp infectivity. (A) Plasmids encoding E2 mutants in the context of an E1E2 polyprotein were used to generate HCVpp (E1N1 to E1N11). Control experiments in the absence of HCV envelope proteins were also performed (−env). Infection assays with the luciferase reporter gene were performed by using target Huh-7 human hepatocarcinoma cells. Similar inputs of viral particles were used in each experiment, and this was confirmed by comparing the amounts of capsid protein incorporated into HCVpp (see panel B, anti-Gag). The results are expressed as percentages of infectivity. For each mutant, the percentage of infectivity was calculated as the luciferase activity of HCVpp produced with mutant envelope proteins divided by the luciferase activity of HCVpp produced with wild-type E1 and E2 (WT). The results are reported as means ± standard deviations of six independent experiments. Pseudotyped particles produced in the absence of envelope proteins were used as a control. The mean luciferase activity of such pseudotyped particles produced in the absence of envelope proteins represented <2% of the activity measured for HCVpp. The luciferase activities (in relative light units) in a representative experiment were as follows: WT, 4.7 × 10⁵; N1, 2.3 × 10⁵; N2, 1.4 × 10³; N3, 4.6 × 10⁵; N4, 1.9 × 10⁴; N5, 2.5 × 10⁵; N6, 2.7 × 10⁵; N7, 4.7 × 10⁵; N8, 4.3 × 10³; N9, 4.7 × 10⁵; N10, 1.4 × 10³; and N11, 1.3 × 10⁵. (B) Incorporation of HCV envelope proteins into HCVpp. Particles were pelleted through 30% sucrose cushions and analyzed by Western blotting. HCV envelope glycoproteins and the capsid protein of MLV were revealed with the following specific MAbs: anti-E1 (A4), anti-E2 (3/11), and anti-MLV capsid (R187, anti-Gag). The expression of mutant proteins was verified by direct Western blotting of cell lysates.