Increased oxidative damage is the likely culprit for the observed mitochondrial degeneration and reduced mitochondrial number in HCV protein-expressing cells. To determine the extent of ROS generation in the mitochondria, MitoSOX was used for live cell staining. The results showed a significant increase in ROS production in the mitochondria across all three cell lines ( Figures 2C and 2D ). After adjusting to the average number of mitochondria per cell, the level of MitoSOX intensity was similar among genome-length replicon, subgenomic replicon, and Coreon cells. Cytosolic and mitochondrial aconitases (ACO1 and ACO2) are very sensitive to inactivation by reactive oxygen and nitrogen species, and reduction in aconitase protein levels and enzyme activities have been used as indicators for increased oxidative stress in their respective subcellular compartments [24, 25] . Our studies showed that ACO1 existed at low levels, whereas ACO2 was barely detectable by western blot analysis, in Huh7 and HCV protein-expressing cells. ACO1 protein level was reduced by 18% in cells with the genome-length replicon ( Figure  S3A ), and total aconitase activity was reduced by 5-23% ( Figure  S3B ) in genome-length and subgenomic replicon cells. The reduction in total aconitase activity most likely reflected increased oxidative stress in the cytosol because the majority of aconitase in Huh7 cells was in cytosolic form.