The HCV sequences of final maxipreps (Qiagen, Valencia, CA) were confirmed (Macrogen Inc., Seoul, Korea). Culturing of Huh7.5 hepatoma cells was as described.[19] 24h before transfection or infection, 4 × 105 cells per well were plated in six-well plates (Nunc; Thermo Fisher Scientific Inc., Waltham, MA). Plasmids were linearized with XbaI, followed by in vitro transcription with T7 RNA polymerase (Promega, Madison, WI) for 2 hours at 37°C. For transfections, 2.5 μg of RNA were incubated with 5 μL of Lipofectamine 2000 in 500 HDAC inhibitor μL of OptiMEM (Invitrogen) for 20 minutes. Cells were incubated with RNA/Lipofectamine complexes

for 16-24 hours. For infections, cells were inoculated with filtered virus-containing

culture supernatant for 16-24 hours. Cultures were evaluated by immunostaining with NS5A Ab 9E10.[19] HCV RNA titers were determined by TaqMan.[19] HCV infectivity titers were determined by adding 10-fold dilutions (starting at 1:2) of supernatants, in triplicate, into 6 × 103 Huh7.5 cells/well of poly-D-lysine-coated 96-well plates (Nunc; Thermo Fisher Scientific). After 48-hour incubation, cells were fixed and immunostained with 9E10 Ab. The number of focus-forming units (ffu) was determined Tamoxifen solubility dmso using an ImmunoSpot series 5 UV analyzer (CTL Europe GmbH, Bonn, Germany).[17, 21, 28] Procedures to generate amplicons for direct sequencing of the complete open reading frame (ORF) and primers for the JFH1 portion were previously reported[19]; Core-NS2-specific

primers are shown in Supporting Table 1. Sequences were analyzed using Sequencher (Gene Codes) and Vector NTI (Invitrogen). Phylogenetic trees were generated using the Jukes-Cantor model and the Neighbor-joining algorithm implemented by Molecular Evolutionary Genetics Analysis (MEGA) software. We analyzed two panels of chronic-phase sera Acesulfame Potassium from HCV genotype 2 patients originating from the Hospital Clinic (Barcelona, Spain) and the National Institutes of Health (Bethesda, MD). All patients were presumably HCV monoexposed, according to clinical records. The genotype and subtype of the infecting HCV was determined by direct sequencing of Core-E1 amplicons[29]; analysis of sample K1118 required cloning of the amplicon. For phylogenetic analysis, we used MEGA. Heat-inactivated (56°C for 30 minutes) patient sera were tested in 2-fold dilutions against J6/JFH1, T9/JFH1, DH8/JFH1, DH10/JFH1, J8/JFH1, and S83/JFH1 and in 5-fold dilutions against J6/JFH1ΔHVR1 and J8/JFH1ΔHVR1.[16] Polyclonal immunoglobulin G (IgG) was purified from 100 μL of serum from four selected samples, using a Protein G HP SpinTrap/Ab Spin Trap system (GE Healthcare, Little Chalfont, UK), and tested against J6/JFH1 and J6/JFH1ΔHVR1 in 5-fold dilutions starting at 100 μg/mL.