Quantifying HCV Replication Levels

We used four HCV subgenomic replicon cell lines, FLR3-1 (genotype 1b, Con-1)47 (link), R6FLR-N (genotype 1b, strain N)26 (link), JFH-1/FLR/K4 (genotype 2a)48 (link), and RMT-tri (genotype 1a)49 (link), which have the firefly luciferase gene for the sensitive and precise quantification of the HCV replication levels using a luciferase assay. We also used REF cells30 (link) which harbor the divided-full genome replicon for analysis of the HCV 5′ UTR sequences. Each cell line was seeded at a density of 5 × 10³ per well in 96-well tissue culture plates, and grown (at 37°C and 5% CO₂) in complete Dulbecco's modified Eagle's medium supplemented with Glutamax I (Invitrogen, Carlsbad, CA) and containing 5% fetal calf serum (Invitrogen). Cells were transfected with 30 nM siRNA using RNAiMax (Invitrogen, Carlsbad, CA). After 72 hours, luciferase activity was determined in triplicate using the Steady-Glo or Bright-Glo luciferase assay kit (Promega Madison, WI). The luciferase signal was measured using an LB940 luminometer (Berthold, Freiburg, Germany) and the results were expressed as the mean percentage of control. IC₅₀ values of siRNA were calculated by nonlinear curve-fitting using the equation: Y = 100-(Y_Bottom × X/(IC₅₀ + X)), where Y represents percent inhibition and X represents the concentration of siRNAs.