Multiple studies in recent years have shown that HCV infection of the human hepatoma cell line Huh7 and immortalized human hepatocytes induce autophagocytic vacuoles and that Trichostatin A HDAC inhibitor autophagy machinery is required for the initiation of HCV Silmitasertib inquirer replication and the production of infectious particles. HCV induces the accumulation of autophagosomes via activation of the unfolded protein response pathway without enhancing autophagocytic protein degradation. Consequently, inhibition of autophagy inhibits HCV replication. Although most studies are carried out with HCV genotype 2a, it is now well accepted that autophagy probably also occurs with infection of other HCV genotypes. What is not clear from these studies is whether enhanced autophagy persists during the chronic phase of HCV infection, and if and to what extent oxidative stress contributes to HCV-mediated activation of autophagy. In this study, we observed the accumulation of autophagocytic vacuoles and up-regulation of the autophagosome marker, LC3, in the genome-length and subgenomic replicon cells. The data suggests that HCV non-structural proteins play a role in inducing autophagy, possibly through ER stress, mitochondrial damage, and induction of oxidative stress. Core-on cells, on the other hand, had a slight increase in the number of autophagocytic vacuoles without a significant increase in LC3-II or total LC3 levels. 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, whereas increased oxidative stress led to a further increase in total LC3 and LC3-II. 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, H2O2 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. Despite up-regulation of multiple antioxidant enzymes in subgenomic replicon cells, 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.