Hepatitis C virus entry depends on clathrin-mediated endocytosis

Abstract

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.

FIG. 1.

PCR-based HCVpp entry assay. (A) PLC/PRF/5 cells were pretreated for 30 min with 20, 50, or 100 nM bafilomycin A1 or left untreated. The cells were then infected with HCVpp, VSV-Gpp, or A-MLVpp in the presence of the drug. After 2 h of spinoculation and an additional hour of incubation, total DNA was purified and retroviral DNA was quantified by real-time PCR. Mean values for the controls with no drug were 1.2 equivalent genomes per cell (EG/cell) for HCVpp, 1.5 EG/cell for VSV-Gpp, and 1.3 EG/cell for A-MLVpp infections. The background value obtained from cells infected with pseudotyped particles produced in the absence of envelope proteins corresponded to 0.03 EG/cell. Results were expressed as the percentage of entry in control cells treated with no drug. (B) PLC/PRF/5 cells were infected by spinoculation with different dilutions of the same HCVpp stock. Cells were either immediately lysed to extract DNA or cultured for 3 days to allow for luciferase expression. Retroviral DNA was quantified by real-time PCR (MLV DNA), and infection was quantified by luciferase assay (Luc). Mean values for the undiluted HCVpp infection were 1.3 EG/cell and 6.5 × 10⁶ relative light units (RLU), and the background values obtained from cells infected with pseudotyped particles produced in the absence of envelope proteins corresponded to 0.018 EG/cell and 3.0 × 10² RLU. Results were expressed as the percentage of entry in cells infected with undiluted HCVpp stock (1:1).

FIG. 2.

Clathrin-mediated entry of HCVpp. (A) PLC/PRF/5 cells were pretreated for 30 min with 2 or 5 μg/ml chlorpromazine (CPZ) or were left untreated. The cells were then infected with HCVpp, VSV-Gpp, or A-MLVpp by spinoculation in the presence of the drug. (B) PLC/PRF/5 cells were transfected twice with CHC siRNA or control siRNA, as explained in Materials and Methods. Two days after the second siRNA transfection, cells were spinoculated with HCVpp, VSV-Gpp, HA/NApp, RD114pp, or A-MLVpp. The cellular content of retroviral DNA was quantified by real-time PCR as a measure of entry and is expressed as a percentage of entry in untreated control cells (A) or in cells treated with control siRNA (B). The inset in panel B shows an immunoblot of the relative CHC and actin contents in siRNA-treated cells.

FIG. 3.

Clathrin-mediated entry of HCVcc. (A to C) Huh-7 cells were treated with CHC or control siRNA and then infected with HCVcc, a recombinant Sindbis virus expressing HCV E1, or SV40. (A) Cells were lysed at 5 hpi (Sindbis) or 30 hpi (HCVcc and SV40), and the cell lysates were analyzed by immunoblotting with antibodies to E2, NS3 (HCVcc), E1 (Sindbis), T-antigen (SV40), CHC, and actin. (B) Cells were fixed and processed for immunofluorescent detection of E2 (HCVcc), E1 (Sindbis), or T antigen (SV40). (B) For each virus, the fields presented contain similar numbers of CHC or control siRNA-treated cells. (C) Percentage of infected cells in CHC and control siRNA-treated cells. (D and E) Huh-7 cells were pretreated for 30 min and then infected for 2 h with HCVcc in the presence or the absence of chlorpromazine (CPZ; 5 μg/ml). Cells infected in the absence of drug were cultured with no drug, or chlorpromazine was added 2 hpi (CPZ 2hpi). Infection was analyzed (D) by immunoblotting with antibodies to E2, NS3, GFP (SeV), and actin or (E) by immunofluorescent detection of infected cells using an anti-E2 MAb (HCVcc) or by GFP fluorescence (SeV). Results are presented as percentages of infected cells.

FIG. 4.

pH dependency of HCVcc infection. Huh-7 cells were pretreated for 30 min and then infected with HCVcc or SeV in the presence of 50 nM bafilomycin A1 or 20 μM chloroquine or in the absence of any inhibitor of endosomal acidification. Cells infected in the absence of drug were cultured with no drug, or bafilomycin A1 was added 2 hpi (Bafilo 2hpi). HCVcc infection was analyzed (A) by immunoblotting with antibodies to E2, NS3, GFP (SeV) and actin or (B) by immunofluorescent detection of infected cells using an anti-E2 MAb (HCVcc) or by GFP fluorescence (SeV). Results are presented as percentages of infected cells.