Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry

Abstract

The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.

FIG. 1.

Frequency of basic residues at each HVR1 position. (A) Frequency in any genotype calculated from the nonredundant 1,489 HVR1 sequences of known genotypes in HCV databases. (B) Frequency in genotype 1a (469 sequences). (C) Frequency in genotype 1b (475 sequences). The height of the box in each bar indicates the frequency of histidine (white box), lysine (gray box), and arginine (black box). The frequency of the basic residues at each position was calculated by dividing the number of basic residues by the total number of sequences.

FIG. 2.

Natural variants of HVR1 modulate HCVpp infectivity. (A) Plasmids encoding different mutants of HVR1 in the context of a full-length E2 glycoprotein were used to generate HCVpp. Control experiments in the absence of HCV envelope proteins were performed (−env). Infection assays with the luciferase reporter gene were performed 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 mean luciferase value of HCVpp containing wild-type proteins was 3.8 × 10⁵ relative light units. Results are reported as the means ± standard deviations of 10 independent experiments. (B) Incorporation of HCV envelope proteins into HCVpp (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 specific anti-E1 (A4), anti-E2 (H47), and anti-MLV capsid (R187 and αGag) MAbs. Expression of mutant proteins was verified by a direct Western blotting on cell lysates (Cells).

FIG. 3.

Role of basic residues of HVR1 in HCVpp infectivity. (A) Plasmids encoding different mutants of HVR1 in the context of a full-length E2 glycoprotein were used to generate HCVpp. Control experiments in the absence of HCV envelope proteins were performed (−env). Infection assays with the luciferase reporter gene were performed 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, αGag). 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 mean luciferase value of HCVpp containing wild-type proteins was 1.6 × 10⁶ relative light units. Results are reported as the means ± standard deviations of 10 independent experiments. (B) Incorporation of HCV envelope proteins into HCVpp (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 specific anti-E1 (A4), anti-E2 (H47), and anti-MLV capsid (R187 and αGag) MAbs. Expression of mutant proteins was verified by direct Western blotting on cell lysates (Cells).

FIG. 4.

Interaction between E2 and heparin. Heparin-Sepharose beads were incubated with lysates of cells expressing HCV envelope glycoproteins (cell) or with HCVpp (pp) lysates. Interaction between E2 and heparin (heparin binding) was revealed by pull-down followed by Western blotting with an anti-E2 MAb (H47). Lysates of HCVpp (HCVpp) and of cells expressing HCV envelope glycoproteins (cell lysate) were analyzed by Western blotting with an anti-E2 MAb (H47) to determine the level of expression of E2 used for heparin binding studies. WT, wild type.

FIG. 5.

Interaction between E2 and CD81-LEL in the context of HVR1 mutants containing different basic motifs. Lysates of HCVpp were incubated with human (h) or murine (m) CD81-LEL (GST fusion protein). Interaction between E2 and CD81-LEL (CD81 binding) was revealed by pull-down with glutathione-Sepharose beads followed by Western blotting with an anti-E2 MAb (H47). Lysates of HCVpp (HCVpp) were analyzed by Western blotting with an anti-E2 MAb (H47) to determine the level of expression of E2 used for CD81 binding studies. Pseudotyped particles or cell lysates produced in the absence of HCV envelope glycoproteins were used as controls (−env). Mutant ΔHVR1 contains an E2 protein deleted of HVR1 sequence. WT, wild type.

FIG. 6.

Role of HVR1 basic residues on HDL-mediated enhancement of HCVpp infectivity. Plasmids encoding different mutants of HVR1 in the context of a full-length E2 glycoprotein were used to generate HCVpp. HCVpp were incubated with Huh7 cells in the presence (30 μg/ml) or in the absence of HDL for 3 h at 37°C. Culture medium was then replaced by 4% lipoprotein-depleted serum. The luciferase activity was analyzed 3 days after inoculation. Similar inputs of viral particles were used in each experiment, and this was confirmed by comparing the amounts of capsid protein incorporated into HCVpp (data not shown). 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). Results are reported as the mean of four independent experiments. Standard deviations were usually lower than 10% and were never above 16%.