Article Figures & Data
Figures
- FIG.1.
Characterization of FL-J6/JFH-5′C19Rluc2AUbi. (A) Schematic of HCV and FL-J6/JFH-5′C19Rluc2AUbi genomes. The 5′ nontranslated region contains an internal ribosome entry site (IRES), which allows translation of the HCV polyprotein (shaded boxes) containing structural (C, E1, E2) and nonstructural proteins (p7, NS2, NS4A, NS4B, NS5A, and NS5B). FL-J6/JFH-5′C19Rluc2AUbi is a full-length HCV genome encoding Rluc, a foot-and-mouth disease virus 2A sequence, and a Ubi sequence. The N terminus of Rluc is fused to the first 19 residues of the HCV C protein (see Materials and Methods). (B) Huh-7.5 cells were infected with FL-J6/JFH-5′C19Rluc2AUbi for 0, 4, 6, 8, 10, 12, 20, 24, 30, 36, or 48 h. At each time point, cells were harvested and luciferase activity was determined. Each point is the average value of triplicate wells; error bars show standard deviations. The dashed line indicates the background level of the assay from naive Huh-7.5 cells. RLU, relative light units.
- FIG. 2.
HCV entry is sensitive to bafilomycin A1 (Baf). (A) Huh-7.5 cells were incubated with bafilomycin A1 (0, 25, 50, or 100 nM) either prior to infection (squares) or at 3 h p.i. (triangles) with HCV. Samples were harvested for luciferase assays at 24 h p.i. Each point is the average value of triplicate wells; error bars show standard deviations. The dashed line indicates the background level of the assay from naive Huh-7.5 cells. (B) Huh-7.5 cells were incubated with bafilomycin A1 (0, 25, 50, or 100 nM) either prior to infection (squares) or at 3 h p.i. (triangles) with HSV-1. Samples were harvested for luciferase assays at 12 h p.i. Each point is the average value of triplicate wells; error bars show standard deviations. (C) Huh-7.5 cells were untreated or incubated with bafilomycin A1 prior to infection (−3 h), at the time of infection (−2 h), or at 0, 1, 2, 3, 4, 5, or 6 h p.i. with HCV. Samples were harvested for luciferase assays at 24 h p.i. Values are expressed as percentages of untreated RLU and are the combined data from two independent experiments done in triplicate; error bars represent standard errors of the means.
- FIG. 3.
HCV entry is sensitive to concanamycin A (Con A) and NH4Cl. (A) Huh-7.5 cells were incubated with concanamycin A (0, 12.5, 25, or 50 nM) prior to infection (squares) or at 3 h p.i. (triangles) with HCV. Samples were harvested for luciferase assays at 24 h p.i. The dashed line indicates the background level of the assay from naive Huh-7.5 cells. (B) Huh-7.5 cells were incubated with concanamycin A (0, 12.5, 25, or 50 nM) prior to infection (squares) or at 3 h p.i. (triangles) with HSV-1. Samples were harvested for luciferase assays at 12 h p.i. (C) Huh-7.5 cells were untreated (0 nM) or incubated with 25 mM NH4Cl prior to infection with HCV (black bars) or HSV-1 (white bars). Samples were harvested for luciferase assays at 24 h p.i. In panels A, B, and C, each point or bar is the average value of triplicate wells; error bars show standard deviations.
- FIG. 4.
HCV infectivity is resistant to acidic pH. (A) HCV or SIN was diluted in citric acid buffer (15 mM citric acid, 150 mM NaCl) at pH 7 (white bars) or pH 5 (black bars) for 10 min at 37°C. Samples were then neutralized and used to infect Huh-7.5 cells. Samples were harvested at 24 h (SIN) or 48 h (HCV) for luciferase assays. Values, expressed as percentages of pH 7 RLU, are the combined data from two independent experiments done in triplicate; error bars represent standard errors of the means. (B) Huh-7.5 cells were treated with bafilomycin A1 (+Baf) (25 nM) and then were infected with HCV (black bars) or SIN (white bars) at 4°C for 2 h. Cells were washed to remove unbound virus and then washed with citric acid buffer at pH 7 or 5. Cells were incubated in the presence of bafilomycin A1 for 24 h when they were harvested for luciferase assays. Luciferase activity from untreated cells washed with the pH 7 buffer (−Baf/pH 7) was expressed as 100 percent. Values are the combined data from two independent experiments done in triplicate; error bars represent standard errors of the means.
- FIG. 5.
HCV is unaffected by inhibitors of CatB and CatL. Huh-7.5 cells were pretreated with CA-074 (100 μM), a CatB selective inhibitor, or FYdmk (0.1 μM), a CatB/CatL inhibitor, and were then infected with HCV (black bars), HIV-VSV-G (gray bars), or VSV-EboV GP (white bars) for 2 h at 37°C. Cells were incubated in the presence of inhibitor for 24 h. At 24 h p.i., cells were harvested for luciferase assays (HCV, HIV-VSV-G) or fluorescence-activated cell sorter analysis (VSV-EboV GP). The luciferase activity or number of green fluorescent protein (GFP)-positive cells from untreated cells is expressed as 100 percent. Each bar is the average value of triplicate wells; error bars show standard deviations.
- FIG. 6.
Incubation at 37°C allows HCV to enter bafilomycin-treated cells. Huh-7.5 cells were treated with bafilomycin A1 (+Baf) (25 nM) and then infected with HCV at 4°C for 2 h. Cells were washed to remove unbound virus and then washed with citric acid buffer at pH 7 (white bars) or 5 (black bars) immediately (0 h p.i.) or after 1 h at 37°C (1 h p.i.). Cells were incubated in the presence of bafilomycin A1 for 24 h when they were harvested for luciferase assays. Luciferase activity from untreated cells washed with pH 7 buffer (−Baf) was expressed as 100 percent. Values are the combined data from two independent experiments done in triplicate; error bars represent standard errors of the means.
