Expression level of the reporter gene, secreted alkaline phosphatase (SEAP), was used to monitor the expression of the HCV genome-length and subgenomic replicons. Huh7 cells with the genome-length or subgenomic replicon were cultured to ,80% confluency and cell culture medium from each cell line was removed for SEAP assays. The SEAP reporter assay was performed using the Great EscAPe SEAP Fluorescence Detection Kit (Clontech), and SEAP activities were normalized to total cellular proteins. To determine total aconitase activities, Huh7 and HCV protein-expressing cells were cultured to 90% confluency in 60 mm plates. Cell pellets were resuspended in aconitase buffer (50 mM Tris-Cl, pH 8, 2 mM citric acid, and 0.6 mM MnCl 2 ) and passed through two rounds of freeze-thaw cycle between liquid nitrogen and room temperature water to break open membranes. Due to the low level of aconitases in Huh7 cells, total aconitase activities were determined with a kinetic assay [24] coupled to the PMS/MTT color reaction to enhance the sensitivity [25] . Following a 3-minute preincubation at 37uC in the dark, the reaction was continued at 37uC and the OD change was monitored every 5 minutes at 590 nm for up to 60 minutes using a plate reader (SpectraMax M3, Molecular Devices, Mountain View, CA, USA). The reaction appeared to be most linear in the first 30 minutes and consequently, OD change per minute, as a function of enzyme activities, was calculated for that time period. The final results were normalized to the amount of total proteins in the cell lysates.