HCV-mediated oxidative stress was likely a contributor in the activation of autophagy because enhanced antioxidant capacity significantly reduced total LC3 and LC3-II levels ( Figure 4A ), whereas increased oxidative stress led to a further increase in total LC3 and LC3-II ( Figure 4B ). ROS generated in the cytosol and mitochondria was equally capable of enhancing autophagy because increased antioxidant capacity in either compartment achieved comparable levels of reduction. In addition, H 2 O 2 appeared to be the main culprit in ROS-mediated activation of autophagy within this experimental system since addition of catalase effectively suppressed X/XO-mediated activation of autophagy ( Figure 4B ). Despite up-regulation of multiple antioxidant enzymes in subgenomic replicon cells ( Figure 5 ), these cells responded to further increase in antioxidant enzymes with reduced autophagy. It is important to note that although the antioxidant enzymes clearly can modulate the effects of viral proteins, they only serve to reduce the LC3 increase by ,26% in subgenomic replicon cells. The data suggest either that other mechanisms, such as ER stress, are important as well or that the antioxidant effect is incomplete. Overexpression of antioxidant enzymes also led to a reduction of autophagy in Core-on and Core-off cells. Antioxidant enzyme overexpression in cells with genome-length replicon, on the other hand, failed to reduce the level of autophagy even though the enhanced antioxidant capacity from SOD/CAT expression vectors was comparable to that of other cells ( Figure  S4 ). In contrast, increased oxidative stress effectively increased autophagy in genome-length replicon cells ( Figure 4B ). The data suggest that oxidative stress may not play a significant role in the basal level of autophagy in genome-length replicon cells; however, the cells are sensitive to additional oxidative stress and are capable of accumulating more autophagosomes under conditions of increased oxidative stress. Although total LC3 levels were increased in X/XO-treated subgenomic replicon cells, LC3-II levels were not changed with the added oxidative stress ( Figure 4B ). It is possible that the conversion of LC3-I to LC3-II was already at the maximum level in subgenomic replicon cells, and additional oxidative stress was not able to enhance the reaction further.