To generate HCVcc expressing Renilla luciferase, we used the GPCR Compound Library screening FL-J6/JFH-5′C19Rluc2AUbi genome [67] kindly provided by C.M. Rice (The Rockfeller University, New York). We replaced the region encoding the J6/JFH-1 HCV polyprotein with the CS-N6 JFH-1 sequence [27]. HCVcc were produced as previously described [7, 27, 67]. HCVcc were added to Huh-7 cells seeded the day before and incubated for 2 h at 37°C. The supernatants were then removed and the cells were incubated in DMEM 10% FBS at 37°C. At 40–48 h post-infection, cells were lysed and processed to measure the Renilla luciferase activities as indicated by the manufacturer (Promega). Luciferase activities were normalized for protein concentration

in each cell lysate. In each figure, results are reported as the mean ± S.D. of three independent experiments. Generation of R1 cell population and resistant cellular clones Huh-7 cells were infected (m.o.i. = 1) with JFH-1/I2/CS-N6 particles [27] 4 h at 37°C

and then maintained for several weeks. Survival cells were amplified and treated with 200 UI/ml of IFN α. After six successive treatments with IFN α, cells were analysed by immunofluorescence and western blotting and subcloned by limiting dilution. The cells were seeded in 96-well plates at 1 cell/well in DMEM 10% FCS. Individual cell clones were amplified and named Huh-7w with a number corresponding to the clone. Cell transfection Huh-7w7 cells were transfected using ExGen500 (Eurogentec) with plasmids expressing human CD81 (pcDNA3.1/hCD81), murine CD81 (pcDNA3.1/mCD81) [30] or the empty vector. Polyclonal populations were obtained by selection for 4 weeks with 600 μg/ml Ulixertinib order of Neomycin (Invitrogen). Antibody neutralization assay Neutralization assays were performed by co-incubating HCVcc/HCVpp and antibodies with target cells 3 h at 37°C. Cells were further incubated for 48 h with DMEM 10% FCS before

measuring the luciferase activities. Cholesterol depletion/replenishment and sphingomyelinase (Smase) treatment Cholesterol depletion was carried out by incubating cells with different concentrations 2-hydroxyphytanoyl-CoA lyase of methyl-β-cyclodextrin (MβCD, Sigma) in serum-free medium at 37°C for 20 min. Cholesterol replenishment of cholesterol-depleted cells was achieved by incubating cells with 1:10 (mol/mol) complex of cholesterol and MβCD (cholesterol water soluble, Sigma) using a 2.5 mM final cholesterol concentration in serum-free medium at 37°C for 15 min. Cholesterol levels in MβCD-treated cells were determined using Amplex Red Cholesterol Assay kit (Molecular Probes). Smase treatments were performed as previously described [47]. Production of HCVpp and infection assays HCVpp were produced as described previously [3, 68] with plasmids kindly provided by B. Bartosch and F.L. Cosset (INSERM U412, Lyon, France). The plasmids encoding HCV envelope glycoproteins of genotypes 1b (UKN1B-5.23), 2b (UKN2B-1.1), 3a (UKN3A-1.28) and 4 (UKN4-11.