Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), glutaMAX-I, and goat, horse, MK-2206 solubility dmso and fetal calf sera were purchased from Invitrogen (Carlsbad, CA). 4′,6-Diamidino-2-phenylindole (DAPI) was from Molecular Probes (Invitrogen). EGCG was from Calbiochem (Merck Chemicals, Darmstadt, Germany), except when a set of green tea catechins, (+)-catechin, (−)-epicatechin (EC), (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and EGCG, was used, which was purchased from Extrasynthèse (Lyon, France).

Stocks were resuspended in dimethyl sulfoxide (DMSO) at 0.5 M. Other chemicals were from Sigma-Aldrich (St. Louis, MO). Mouse anti-E1 A4, 16 rat anti-E2 3/11, 17 mouse anti–yellow fever virus (YFV) envelope protein 2D12 (ATCC CRL-1689),

and mouse anti–bovine viral diarrhea virus (BVDV) NS3 Osc-23 18 monoclonal antibodies Selleck Acalabrutinib (mAbs) were produced in vitro. Cyanin 3 (Cy3)-conjugated goat antimouse immunoglobulin G (IgG) was from Jackson Immunoresearch (West Grove, PA). Huh-7, 19 HEK 293T (ATCC number CRL-11268), Vero (ATCC CCL-81), and Madin-Darby Bovine Kidney (MDBK; ATCC number CCL-22) cells were grown in DMEM, supplemented with glutaMAX-I and either 10% fetal calf serum (Huh-7, HEK

293T, and Vero) or 10% horse serum (MDBK). We used a modified Japanese fulminant hepatitis (JFH)1 virus containing titer-enhancing mutations, 20 in which the A4 epitope of HCV glycoprotein E1 of genotype 1a was reconstituted. 21 The JFH1-Luc plasmid, containing a Renilla Luciferase reporter gene, the JFH1-ΔE1/E2-Luc or JFH1-ΔE1/E2 clonidine plasmids, which contain an in-frame deletion in the E1/E2 region, and the JFH1/GND-Luc replication mutant, have been described previously. 21, 22 Infections were scored by measuring luciferase activity in cell lysates, using a Renilla luciferase assay system from Promega (Madison, WI), or by measuring infectivity by indirect immunofluorescence (IF) with anti-E1 mAb. For quantitative binding experiments, purified virus was obtained by the precipitation of HCV grown in cell culture (HCVcc)-infected Huh-7 cell supernatants with 8% polyethylene glycol 6000. Pelleted virus was then loaded onto a continuous 10%-40% iodixanol gradient.