Article Figures & Data
Figures
- FIG. 1.
Anti-CD81 MAb inhibits HCV pseudotype infection of Huh-7.5, Hep3B, and PLC/PR5 cells. Huh-7.5, Hep3B, and PLC/PR5 cells were incubated with anti-CD81 MAb (5A6) or an irrelevant isotype-matched IgG at 5 μg/ml and infected with pseudotypes bearing HCV H or Con1 gp's. Viral stocks were normalized for p24 HIV core antigen and used for infection at 1 ng/well. All infections were performed in triplicate, and the mean luciferase activity (in relative light units [RLU]) is shown. In this and subsequent figures, some standard deviation bars for RLU values are not visible due to compression from the log scale used, but all are within 10%.
- FIG. 2.
siRNA silencing of CD81 expression in Huh-7.5 cells inhibits HCV pseudotype infection. (A) Huh-7.5 cells transfected with either siIRR or siCD81 were stained with IgG1 or anti-CD81 MAb (1.3.3.22) at 54 h posttransfection and analyzed by flow cytometry. (B) siRNA-transfected Huh-7.5 cells were infected with the indicated pseudotypes and analyzed for luciferase activity. (C) siRNA-transfected Huh-7.5 cells were infected similarly with the indicated range of HIV-HCV H pseudotype inocula and analyzed for luciferase activity. All infections were performed in triplicate, and the mean luciferase activity is shown.
- FIG. 3.
Expression of CD81 in HepG2 and HH29 cells confers susceptibility to HCV pseudotype infection. (A) HepG2, HH29, and Huh-7.5 cells alone (upper panels) or after transduction with TRIP-CD81 (lower panels) were stained with an irrelevant IgG and anti-CD81 MAb (1.3.3.22) and analyzed for CD81 expression by flow cytometry. Quadrants were established based on irrelevant isotype-matched IgG staining of the cells. FSC, forward scatter. (B) Transduced (shaded bars) and nontransduced (hatched bars) HepG2, HH29, and Huh-7.5 cells were infected with the indicated pseudotypes 48 h posttransduction and analyzed for luciferase activity. (C) Transduced cells were incubated with either MAbs specific for CD81 (5A6) or an irrelevant isotype-matched IgG at 5 μg/ml and infected with HIV-HCV H E1E2 or with HIV-HCV H E1E2 preincubated with anti-E2 MAb 11/20. All infections were performed in triplicate, and the mean luciferase activity is shown.
- FIG. 4.
The LEL of CD81 is the essential structural component for conferring CD81-dependent susceptibility to HCV pseudotype infection. (A) Schematic representation of CD81 (solid line), CD9 (dotted line), the chimeric molecules CD81-9LEL and CD9-81LEL, and the CD81 mutants with point mutations. (B) HepG2 cells expressing wild-type CD81 (CD81wt) and CD81 variants were infected with the indicated pseudotypes and analyzed for luciferase activity. All infections were performed in triplicate, and the mean luciferase activity is shown. CD9wt, wild-type CD9. (C) Binding of strain H and Con1 soluble E2 to HepG2 cells transduced to express wild-type CD81 and CD81 variants. Results are expressed as median fluorescence intensities. RFU, relative fluorescence units.
Tables
- TABLE 1.
Susceptibility of cells to HCV pseudotype infection
Cell line Cell type Human CD81 expressiona Infectivity (RLU, 103) of pseudotypes expressing: No Env VSV G SF162 H E1E2 Con1 E1E2 Hos.CD4.R5 Human osteosarcoma 24.5 0.1 950.4 120.0 0.1 0.1 Huh-7 Human hepatoma 48.0 0.1 4,986.9 0.8 68.6 58.3 Huh-7.5 Human hepatoma 81.3 0.1 5,469.3 0.1 89.3 63.2 HepG2 Human hepatoma 1.9 0.1 890.0 0.9 0.1 0.1 PLC/PR5 Human hepatoma 108.0 0.1 3,553.3 0.1 9.4 7.5 HepH Human liver 67.5 0.1 218.7 0.1 0.1 0.1 HH29 Human liver 2.2 0.1 3,031.7 0.1 0.1 0.1 Hep3B Human liver 49.4 0.7 5,123.6 0.1 120.3 89.5 SK Hep1 Human liver 67.9 0.2 3,812.3 0.1 0.1 0.2 Fet Hep1.3 Human liver 99.4 0.2 2,869.5 0.1 0.1 0.2 Hepa1-6 Mouse liver NA 0.1 410.7 0.2 0.1 0.1 H2-35 Mouse liver NA 0.1 232.1 0.2 0.2 0.1 AML 12 Mouse liver NA 0.1 332.6 0.1 0.1 0.1 FT0-2B Rat liver NA 0.1 109.1 0.1 0.1 0.1 PBMC Human peripheral blood, resting NT 0.1 0.1 0.1 0.1 0.1 PBMC Human peripheral blood, phytohemagglutinin activated NT 0.1 0.9 66.3 0.1 0.1 MT-2 Human T lymphoid 95.6 0.1 913.1 0.1 0.1 0.1 Molt 4 Human T lymphoid 67.3 0.2 3.9 0.1 0.1 0.1 Hut-78 Human T lymphoid 171.5 0.1 2.6 0.1 0.1 0.1 Daudi Human B cell 257.3 0.1 589.3 0.1 0.1 0.1 HeLa Human epithelial 114.4 0.1 61.3 0.1 0.1 0.1 293-T Human embryonal kidney 189.4 0.1 1,300.1 0.1 0.1 0.1 SW13 Human colorectal adenocarcinoma 76.4 0.1 559.2 0.2 0.1 0.1 Caco-2 Human colon adenocarcinoma 45.9 0.1 250.3 0.1 0.1 0.1 HT-29 Human fibrosarcoma NT 0.1 1,156.8 0.1 0.1 0.1 ↵ a Median fluorescence intensity, in relative fluorescence units. NA, not applicable; NT, not tested.
