Unknown

HCV replicon RNA-containing Huh7 cells (clone A cells; Apath, LLC, St. Louis, Mo.) were kept at exponential growth in Dulbecco’s modified Eagle’s medium (high glucose, no pyruvate) containing 10% fetal bovine serum, 1x nonessential amino acids, penicillin-streptomycin-glutamine (100 U/ml, 100 mkg/ml, and 0.292 mg/ml, respectively), and G418 (500 to 1,000 mkg/ml). Antiviral assays were performed in the same medium without G418. Cells were seeded in a 96-well plate at 1,000 cells per well, and test compound 9 (RO-2433) were added immediately after seeding. Incubation times differed according to the type of experiment. At the end of the incubation step, total cellular RNA was isolated (RNeasy 96 kit; Qiagen). Replicon RNA and an internal control (Taq- Man rRNA control reagents; Applied Biosystems) were amplified in a single-step multiplex RT-PCR protocol as recommended by the manufacturer.