Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I

Abstract

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.

FIG. 1.

SR-BI is required for JFH-1 infection. Huh-7 cells were incubated with anti-CD81 or anti-SR-BI antibody and inoculated with JFH-1, and infectivity was analyzed by measuring intracellular JFH-1 RNA at 3 days postinfection. (A) Huh-7 cells were infected with JFH-1 in the presence of three dilutions each of anti-CD81 (250, 25, and 10 μg/ml) and anti-SR-BI (1:20, 1:50, and 1:100) antibodies. As controls, Huh-7 cells were incubated with isotype-matched mouse IgG (mIgG) and PI rat serum at 250-μg/ml and 1:50 dilutions, respectively. (B) Huh-7 cells were incubated with concentrations of anti-CD81 antibodies alone (bars with horizontal lines), anti-SR-BI antibodies alone (unfilled bars), or both antibodies (bars with diagonal lines), and intracellular JFH-1 RNA levels were measured 3 days later. Two dilutions each of anti-CD81 (25 and 2.5 μg/ml) and anti-SR-BI (1:50 and 1:100) antibodies were used. To demonstrate cooperativity, Huh-7 cells were incubated with combinations of anti-CD81 and anti-SR-BI antibodies. Cells were incubated with anti-CD81 and anti-SR-BI antibodies (25 μg/ml anti-CD81 plus 1:50 anti-SR-BI and 2.5 μg/ml anti-CD81 plus 1:100 anti-SR-BI). As controls for anti-CD81 and anti-SR-BI antibodies, Huh-7 cells were incubated with equivalent concentrations of isotype-matched mouse IgG and PI rat serum, respectively. HCV RNA copy numbers were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) copy numbers for all samples. These are representative data (means ± SDs) for three independent experiments. (C) Inhibition of HCVpp entry using antibodies specific to CD81 (25 mg/ml) and SR-BI (1:50) individually or in combination. TU, transducing units. These are representative data (means ± SDs) for three replicates.

FIG. 2.

Anti-SR-BI antibodies inhibit JFH-1 NS5A protein expression. Indirect immunofluorescence analysis of NS5A protein expressed in JFH-1-infected Huh-7 cells alone (A) or in the presence of anti-CD81 (25 μg/ml) (D), anti-SR-BI (1:50) (E), or a combination of both anti-CD81 and anti-SR-BI antibodies (F). Huh-7 cells were incubated with equivalent concentrations of isotype-matched mouse IgG (mIgG) (B) and PI rat serum (C) as controls for the anti-CD81 and anti-SR-BI antibodies, respectively. The bars represent 400-μm distances. These are representative data for three independent experiments.

FIG. 3.

MβCD treatment of Huh-7 cells decreases CD81 plasma membrane expression. CD81 expression was analyzed in mock-treated Huh-7 cells (A) or in Huh-7 cells after treatment with 10 mM MβCD (B). CD81 expression was also determined in MβCD-treated cells to which exogenous cholesterol was added (C). SR-BI expression was also analyzed in mock-treated (D) and MβCD-treated (E) Huh-7 cells. The bars represent 60-μm distances. These are representative data for at least two independent experiments.

FIG. 4.

CD81 cell surface expression decreases after MβCD treatment. Cell surface expression of CD81 (A) and SR-BI (B) was analyzed in mock-treated Huh-7 cells (M), Huh-7 cells that were treated with 10 mM MβCD (MβCD), or MβCD-treated cells which were further incubated with cholesterol (MβCD+C). (C) MFI of CD81 surface expression after mock treatment or treatment with 10 mM MβCD in the presence (MβCD+C) or absence (MβCD) of exogenous cholesterol are plotted. These are representative data for at least two independent experiments.

FIG. 5.

Depletion of cholesterol by MβCD decreases JFH-1 infection in Huh-7 cells. Huh-7 cells were untreated or pretreated with 7.5 mM MβCD alone (MβCD) or MβCD plus cholesterol (MβCD + C) prior to infection with JFH-1. (A) Total RNA was harvested 3 days later, and levels of intracellular JFH-1 RNA were quantitated by RT-PCR. Relative HCV levels were measured by normalization to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) copy numbers for each sample. (B) Intracellular cholesterol levels were quantitated in mock-treated Huh-7 cells or cells that were treated with MβCD alone (MβCD) or MβCD plus cholesterol (MβCD + C). (C) Viable cells were quantitated by trypan blue exclusion after MβCD treatment (in the absence or presence of cholesterol) and compared to mock-treated Huh-7 cells.

FIG. 6.

Depletion of cholesterol by MβCD does not affect HCV RNA replication. (A) Huh-7 cells stably replicating the JFH-1 SG replicon were treated with 7.5 mM MβCD, and intracellular levels of JFH-1 RNA were measured at 1 and 2 days posttreatment. (B) Intracellular cholesterol levels in Huh-7 cells were quantitated after treatment with MβCD (MβCD) or MβCD plus cholesterol (MβCD+C). (C and D) Huh-7 cells were infected with JFH-1 for 9 days, after which cells were untreated or treated with 7.5 mM MβCD (MβCD) or MβCD plus cholesterol (MβCD+C). Intracellular JFH-1 RNA levels (C) and JFH-1 virus secretion (D) were quantitated before the start of treatment (day 0) and at 2 days posttreatment. These are representative data (means ± SDs) for three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ffu, focus-forming units.

FIG. 7.

Depletion of cholesterol by MβCD decreases HCVpp entry in Huh-7 cells. (A) Dose-dependent inhibition of HCVpp infection of Huh-7 cells after treatment with 2.5, 5, and 10 mM MβCD. (B) Huh-7 cells were untreated or treated with 10 mM MβCD (MβCD) or 10 mM MβCD plus cholesterol (MβCD+C) and infected with HCVpp or MLVpp. Relative light units (RLU) for each sample were calculated by normalization to mock-treated Huh-7 cells. These are representative data (means ± standard errors of the means) for two independent experiments.