Besides increased ROS production in the mitochondria of all three cell lines analyzed, our data on aconitase protein levels and enzyme activities also suggested an increased ROS production in the cytosol in genome-length and subgenomic replicon cells. The reduction in ACO1 protein level in genome-length replicon cells is reminiscent to that observed in CuZnSOD deficient mice [25] , in which heightened state of oxidative stress in the cytosol may lead to increased degradation of irreversibly inactivated aconitase. Subgenomic replicon cells had reduced aconitase activity without a significant reduction in the protein level ( Figures S3A and S3B) . The data suggests that ACO1 may be reversibly inactivated due to increased oxidative stress in the cytosol. Taken together, our study results suggest that HCV non-structural proteins are capable of causing increased oxidative stress in both the mitochondrial and the cytosolic compartments, whereas the effects of core protein is mainly limited to the mitochondria.