Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies

Abstract

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

FIG. 1.

JFH-1 G451R has an altered dependence on SR-BI. (A) Huh-7.5 cells overexpressing SR-BI were incubated with JFH-1 wt or JFH-1 G451R for 8 h. Cells were fixed after 48 and 72 h and stained for NS5A, and the mean number of infected cells per well was determined. Infectivity is expressed relative to parental Huh-7.5 cells. (B) Huh-7.5 cells were incubated with a serial dilution of rabbit anti-SR-BI serum for 1 h prior to challenge with JFH-1 wt or JFH-1 G451R. The data are expressed as percent neutralization relative to infection of Huh-7.5 cells treated with control rabbit serum. (C) Huh-7.5 cells were inoculated with JFH-1 wt or JFH-1 G451R in the presence of 10 μg/ml HDL. Infection is expressed relative to infection in the absence of HDL. Error bars indicate standard deviation from the mean (n = 3; P = 0.0069, unpaired t test).

FIG. 2.

CD81 dependence of JFH-1 wt and G451R infection. Huh-7.5 cells were incubated with a serial dilution of 2s131 (A) or 1s201 (B) mouse anti-CD81 MAb for 1 h prior to challenge with JFH-1 wt (filled circles) or JFH-1 G451R (open circles). (C) JFH-1 wt or G451R viruses were incubated with human CD81 LEL for 1 h prior to infection of Huh-7.5 cells. The data are expressed as percent neutralization relative to viral infection in the presence of an irrelevant mouse IgG or nonactive mouse CD81 LEL, respectively. Error bars indicate standard deviation from the mean (n = 3).

FIG. 3.

Interaction of JFH-1 wt and G451R sE2 with CHO cells expressing SR-BI and CD81. (A) CHO-SR-BI cells (>80% positive) or CHO-CD81 cells (20 to 30% positive) were methanol fixed and stained with MAb anti-CLA1 or anti-CD81 2s139. (B) Binding of recombinant JFH-1 wt and G451R sE2 to parental CHO cells and CHO-SR-BI and CHO-CD81 cells. Bound E2 was detected with MAb 10/76b. (C) The mean fluorescence intensity (MFI) of JFH-1 wt and G451R sE2 bound to CHO-SR-BI and CHO-CD81 cells is shown; the signal from the sE2-CHO cell interaction was subtracted. Error bars indicate standard deviation from the mean (n = 3).

FIG. 4.

Interaction JFH-1 wt and G451R sE2 with recombinant CD81. (A) Dose-dependent binding of JFH-1 wt (gray bars) or G451R (white bars) sE2 with hCD81 LEL dimer. Data are represented as the mean optical density (OD) at 450 nm. (B) JFH-1 wt or G451R sE2 association with PBS, monomeric and dimeric hCD81 LEL, and mutants of hCD81 LEL that abrogate CD81 interaction with E2. All mutants were characterized for their effects on CD81 oligomerization and were shown to have minimal effect on dimerization. E2 binding to the mutants is expressed relative to CD81 LEL dimer. Error bars indicate standard deviation from the mean (n = 3).

FIG. 5.

Analysis of JFH-1 wt and G451R buoyant density. Concentrated JFH-1 wt and G451R were separated on an iodixanol gradient. (A) The number of HCV particles per fraction was assessed by quantifying HCV RNA genomes by RT-PCR. The particle number in each fraction is expressed as a percentage of the total for either virus. (B) The infectivity per fraction was assessed by inoculating Huh-7.5 cells. The infectivity within each fraction is expressed as a percentage of the total for either virus. Error bars indicate standard deviations from the mean (n = 3). (C) The number of infectious units per RNA genome copy were calculated to analyze the specific infectivity of particles within each fraction.

FIG. 6.

Relationship between particle density and coreceptor dependency. The density fractions containing infectious JFH-1 wt or JFH-1 G451R were used to assess the relationship between particle density and SR-BI and CD81 interaction(s). The approximate densities of each fraction are indicated on the x axis. (A) Huh-7.5 cells overexpressing SR-BI were inoculated with JFH-1 wt and G451R virus, and infectivity is expressed relative to parental Huh-7.5 cells. Huh-7.5 cells were incubated with either anti-SR-BI serum at 1/300 (B) or anti-CD81 1s201 at 0.1 μg/ml (C) prior to challenge with infectious fractions of JFH-1 wt and G451R. Error bars indicate standard deviations from the mean (n = 3).

FIG. 7.

JFH-1 G451R demonstrates an increased sensitivity to neutralization by gp-specific antibodies. JFH-1 wt (closed circles) or G451R (open circles) was incubated with IgG purified from the sera of six HCV-infected subjects (A to F) or anti-E2 MAb 3/11 (H) prior to infection of Huh-7.5 cells. Percent neutralization was calculated by quantifying viral infectivity in the presence of anti-HCV-specific antibodies relative to HCV-negative IgG or irrelevant MAb, respectively. (G) To determine the concentration of IgG required to neutralize 50% of JFH-1 and G451R infectivity (IC₅₀), both viruses were incubated with a pool of the six-patient-derived IgG. The IC₅₀ is depicted as a horizontal line. Error bars indicate standard deviations from the mean (n = 3).

FIG. 8.

Association between JFH-1 particle density and sensitivity to neutralizing antibodies. JFH-1 wt and G451R were separated on an iodixanol gradient as detailed in the legend of Fig. 5, and the fractions were tested for their sensitivity to neutralization by pooled HCV-infected patient IgG (10 μg/ml). Data are expressed as percent neutralization calculated by comparing infectivity in the presence of HCV-negative IgG. A positive correlation was observed between JFH-1 particle density and neutralization by pooled patient IgG (P < 0.0001, unpaired t test). Error bars indicate standard deviations from the mean (n = 4).