Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles

Abstract

Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.

FIG. 1.

Inhibition of viral entry by drugs that target different internalization pathways. Huh7 cells were treated with chlorpromazine (25 μM), filipin (5 μg/ml), or nystatin (12.5 μg/ml) for 1 h at 37°C. The mock treatments were water for chlorpromazine and nystatin as well as methanol for filipin. Cells were infected with HCVpp, SFVpp, VSVpp, or HTLV-1pp in the continued presence of drugs or corresponding mock treatment. Media were changed after 2 h, and luciferase activity in cellular extracts was measured 48 h postinfection. The percent entry was calculated relative to mock-treated cells, and values are means of at least three independent experiments ± standard deviations.

FIG. 2.

Inhibition of pseudoparticle entry by agents that target clathrin-mediated endocytosis. (A) Huh7 cells were lipofected with a nonspecific siRNA (control) or an siRNA targeting the clathrin HC (α-CHC). Seventy-two hours posttransfection, cellular extracts were analyzed for clathrin HC and β-tubulin expression by Western blotting. (B) Huh7 cells were infected with HCVpp or HTLV-1pp 72 h after transfection with siRNAs. Luciferase activity in cell lysates was measured 48 h postinfection. The percent entry was calculated relative to the nonspecific siRNA-transfected cells. Results are from at least three independent experiments ± standard deviations. (C) Huh7 cells were transduced with pQCXIP backbone vector, pQCXIP-EGFP-Eps15(D3Δ2), or pQCXIP-EGFP-Eps15(Δ95/295). Cells were infected with HCVpp, SFVpp, or HTLV-1pp 48 h posttransduction. Luciferase activity in cell lysates was measured 48 h postinfection. The percent entry was calculated relative to the backbone vector-transduced cells. Results are from at least three independent experiments ± standard deviations.

FIG. 3.

Inhibition of pseudoparticle entry by Rab5 and Rab7 transdominant-negative mutants. (A) Huh7 cells were transduced with pQCXIP backbone vector, pQCXIP-EGFP-Rab5(WT), or pQCXIP-EGFP-Rab5(S34N). (B) Alternatively, Huh7 cells were transduced with pQCXIP backbone vector, pQCXIP-EGFP-Rab7(WT), or pQCXIP-EGFP-Rab7(T22N) and infected with viral pseudotypes. Forty-eight hours posttransduction, cells were infected with HCVpp, SFVpp, or HTLV-1pp. Luciferase activity in cell lysates was measured 48 h postinfection. The percent entry was calculated relative to the backbone vector-transduced cells. Results are from at least three independent experiments ± standard deviations. WT, wild type.

FIG. 4.

Time course of proteinase K and bafilomycin A1 sensitivity of viral entry. (A) HCVpp, (B) SFVpp, or (C) VSVpp were prebound to Huh7 cells at 4°C for 1 h. Entry was initiated by shifting cells to 37°C. At the indicated time points, cells were treated with proteinase K (50 μg/ml) at 4°C for 1 h (dotted line). Cells were washed, resuspended in fresh medium, and reseeded. Alternatively, bafilomycin A1 (25 nM) was added at the indicated time points (solid line). After 3 h, the drug was removed by a change of medium. Luciferase activity was measured in cellular extracts 48 h postinfection. The percent entry was calculated relative to the 3-h time point, and values are means of at least three independent experiments ± standard deviations.

FIG. 5.

Rescue of pseudoparticle entry by extracellular low-pH treatment. (A) HTLV-1pp, (B) SFVpp, (C) VSVpp, or (D) HCVpp were prebound to Huh7 cells in the presence of bafilomycin A1 (25 nM) or DMSO (1:1,000) at 4°C for 1 h. Unbound particles were washed off, and internalization was initiated by shifting cells to 37°C. At the indicated time points, cells were incubated for 5 min with DMEM (pH 7.2 or pH 5.3) and then returned to fresh medium supplemented with bafilomycin A1. After 2 h, the drug was removed by a change of medium. Luciferase activity was measured in cellular extracts 48 h postinfection. The percent entry was calculated relative to cells treated with DMSO, and values are means of at least three independent experiments ± standard deviations.