Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain

Abstract

In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

Keywords: envelope proteins; glycoprotein; hepatitis C virus; viral assembly; viral entry.

FIG 1

E1 C-terminal region sequence analyses. (A) The E1 aa270-350 sequence from the HCV JFH1 strain (AB047639; genotype 2a) is indicated with respect to the polyprotein numbering. Amino acids mutated in this study are indicated by a red dot. (B) Amino acid repertoires of the C-terminal region of E1. The amino acid (aa) repertoire was deduced from the ClustalW multiple alignment of the 28 representative E1 sequences from confirmed genotypes and subtypes in the European HCV database (https://euhcvdb.ibcp.fr/euHCVdb/jsp/nomen_tab1.jsp). Amino acids observed at a given position in fewer than two distinct sequences were not included. Amino acids observed at a given position in more than 25 distinct sequences are shown in capital letters. The degree of amino acid conservation at each position can be inferred from the extent of variability (with the observed amino acids listed in decreasing order of frequency from top to bottom), together with the similarity index according to ClustalW convention (asterisk [*], invariant; colon [:], highly similar; dot [.], similar).

FIG 2

Effect of E1 mutations on the expression of viral proteins. Viral RNA transcribed from JFH1-derived mutants was electroporated into Huh-7 cells that were lysed 48 h later. Viral proteins were separated by SDS-PAGE and revealed by Western blotting with MAbs A4 (anti-E1), 3/11 (anti-E2), and anti-NS5A, as well as anti-beta-tubulin antibody, to verify loading of equal amounts of cell lysates. The protein detected by MAb A4 in cells expressing ΔE1E2 mutant corresponds to a fusion protein between the N terminus of E1 and the C terminus of E2.

FIG 3

Effect of mutations on extracellular and intracellular infectivities. Viral RNA transcribed from JFH1-derived mutants was electroporated into Huh-7 cells. The infectivities of the supernatants and intracellular viruses were determined at 48, 72, and 96 h postelectroporation by titration. Error bars indicate standard errors of the means from at least three independent experiments. Values were compared to the wild-type virus. Differences were considered statistically significant for the extracellular infectivity of mutants G278A, D279A, G282A, L286A, Q302A, E303A Y309A, G311A, T314A, G315A, H316A, M318A, W320A, D321A, M322A, W326A, R339A, and P341A (P < 0.05) and for the intracellular infectivity of mutants G278A, D279A, G282A, L286A, Q302A, E303A Y309A, G311A, T314A, G315A, H316A, M318A, W320A, D321A, M322A, R339A, and P341A (P < 0.05) at 96 h postinfection.

FIG 4

Effects of E1 mutations on HCV core protein secretion. Huh-7 cells were electroporated with wild-type or mutant viral RNAs. The levels of core protein in supernatants and cell lysates were determined at 48 h postelectroporation. Error bars indicate standard error of the means from at least three independent experiments. Values of core protein were compared to the wild-type value. Differences were considered statistically significant for extracellular mutants G278A, D279A, G282A, L286A, Q302A, E303A Y309A, and P341A (P < 0.05).

FIG 5

Effect of E1 mutations on E1E2 conformation. (A, upper panel) Interaction of HCV glycoproteins and CD81 (HCV entry factor). E1 and E2 from cell lysates were analyzed by GST pulldown at 48 h postelectroporation using a CD81-LEL-GST fusion protein. Pulled-down E1 and E2 were separated by SDS-PAGE and revealed by Western blotting with MAbs anti-E1 (A4) and anti-E2 (3/11). (A, lower panel, and B) Recognition of HCV E1 and E2 glycoproteins by conformation-sensitive anti-E1E2 MAb AR5A and anti-E1 MAb IGH526, as indicated. At 48 h postelectroporation, E1 and E2 proteins from cell lysates were analyzed by immunoprecipitation with MAbs AR5A and IGH526. Immunoprecipitated proteins were revealed by Western blotting using MAbs A4 and 3/11.

FIG 6

Effect of E1 mutations on E1E2 interaction with HCV neutralizing antibodies and CD81. CD81 inhibition assays (A) and AR5A (B) and AR4A (C) neutralization experiments were carried out by incubating E1 mutants or wild-type virus with increasing concentrations of human CD81-LEL, MAb AR5A, or MAb AR4A at 37°C for 2 h. The mixture was then added to naive Huh-7 cells that were plated 1 day before. At 72 h postinfection, infectivity was determined by immunofluorescence. The values are the combined data from three independent experiments. The error bars represent standard errors of the means. Results were compared to those of the wild type and a P value of <0.05 was obtained for mutants M323A, W326A, P328A, and R339A in the AR5A and AR4A neutralization experiments. (D) Recognition of HCV E1 and E2 glycoproteins by conformation-sensitive anti-E1E2 MAb AR4A. At 48 h postelectroporation, E1 and E2 proteins from cell lysates were analyzed by immunoprecipitation with MAb AR4A. Immunoprecipitated proteins were revealed by Western blotting with MAbs A4 and 3/11.

FIG 7

Effect of E1 mutations on the recognition of HCV receptors. Huh-7 cells were preincubated at 37°C for 2 h with increasing concentrations of antibodies targeting HCV receptors: anti-CD81 MAb JS81 (A), anti-CLDN1 MAb OM8A9-A3 (B), and anti-SR-BI MAb Cla-I (C). E1 mutants or wild-type virus were then inoculated onto the cells. At 72 h postinfection, the residual infectivity was determined by immunofluorescence. The values are the combined data from three independent experiments. The error bars represent standard errors of the means. Results were compared to those of the wild type. A P value of <0.05 was determined for mutants L286A, E303A, M323A, and P328A in the presence of anti-CLDN1 MAbs and for mutants M323A, W326A, P328A, and R339A in the presence of anti-SR-BI MAbs. (D) SRB1 or CD81 expression was downregulated by siRNA targeting SRB1 or CD81 mRNA. Infectivity is expressed as the percentage of infection performed in the presence of the control siRNA. Mean values and standard deviations from three independent experiments are shown. The unpaired t test was used to compare the infectivities of the wild-type and mutant viruses. Differences were considered statistically significant if the P value was <0.05.

FIG 8

Density gradient analyses of SRBI independent mutants. Concentrated supernatants of cells electroporated with HCV RNA were separated by sedimentation through a 10 to 50% iodixanol gradient. Fractions were collected from the top and analyzed for their infectivity by titration and for their density.

FIG 9

Effect of E1 mutations on viral RNA incorporation. Huh-7 cells were electroporated with mutants and wt RNA. (A) At 48 h postelectroporation, intracellular viral RNA was extracted and quantified by quantitative RT-PCR. In parallel, the viral particles were precipitated from the supernatant with polyethylene glycol and concentrated by ultracentrifugation. (B) Extracellular viral RNA contained in the concentrated virus was extracted and quantified by quantitative RT-PCR.

FIG 10

Effect of E1 mutations on HCVpp infectivity. Infectivity of HCVpp harboring E1 with G311A, G315A, or W320A mutation. (A) HCVpp infectivity was determined by measuring the activity of the luciferase reporter gene in infected Huh7 cells. Pseudotyped particles produced in the absence of envelope proteins were used as negative controls. The results are reported as means ± the standard deviations (error bars) of three independent experiments. A P value of <0.05 was obtained for mutants G311A, G315A, and W320A. (B) Effect of E1 mutations on the incorporation of envelope proteins in HCVpp particles. Cells producing HCVpp were lysed and analyzed by Western blotting. HCVpps contained in the supernatants of transfected 293T cells were concentrated on a 20% sucrose cushion by ultracentrifugation and analyzed by Western blotting. E1, E2, and capsid were detected using MAbs A6, 3/11, and CRL1912, respectively.