In summary, our studies using Huh7 cells expressing different parts of the HCV polyprotein suggest that the presence of the fulllength HCV polyprotein leads to the most severe cellular damage, including generation of ROS, accumulation of lipids, ER stress, mitochondrial injury and degeneration, accumulation of autophagosomes, and prolonged population doubling time. Despite the elevated levels of ROS, these cells failed to mount a robust antioxidant response. Huh7 cells with only the non-structural proteins, on the other hand, have the same cytopathic changes as that observed in the genome-length replicon cells, but the cells showed a robust antioxidant response even though the response was not sufficient to suppress HCV-mediated ROS production. Our data suggest that HCV-mediated ER stress, mitochondrial injury, and oxidative stress form the three arms of mediators for the activation of autophagy (Figure 7) . Their effects are additive and inter-related. Without a concomitant increase in downstream protein degradation, autophagosomes accumulate and provide a sanctuary for HCV replication and protection from host immune surveillance [42] . The process likely helps to sustain a low level of viral production, which perpetuates chronic HCV infection and chronic liver injury (Figure 7) . In addition to activation of autophagy, HCV-mediated ER stress can lead to imbalance in Ca homeostasis, which further contributes to cellular oxidative stress [14, 15, 46, 47] . Mitochondrial injury can lead to metabolic deficits Figure 7 . Diagrammatic presentation of the cytopathic effects caused by various HCV proteins. Replication of HCV RNA and production of HCV structural (core, E1, E2, and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) in the rough ER and the subsequent partition of HCV proteins to different subcellular compartments lead to ER stress, mitochondrial injury, and the production of ROS. These cytopathic effects lead to activation of autophagy without a concomitant increase in protein degradation. Consequently, autophagosomes accumulate in HCV infected cells. EM photo with normal mitochondria (M) and rough ER (RER) was taken from healthy Huh7 cells; EM photos with enlarged mitochondria, ER blebbing, lipid droplet (L), and autophagosomes (A) were taken from genome-length and subgenomic replicon cells. The HCV genetic materials contained in genome-length replicon, subgenomic replicon, and Core-on cells are depicted at the top of the diagram. doi:10.1371/journal.pone.0028551.g007 and further exacerbates cell injury and dysfunction. Oxidative stress can cause increased DNA damage and mutation, inactivation of redox-sensitive proteins, and activation of redox sensitive signaling pathways such as MAPK and AP-1 [48] [49] [50] . These cytopathic effects work in concert in the course of chronic HCV infection to promote cell death, hepatocyte turnover, alteration of the liver microenvironment, and ultimately cell transformation and tumorigenesis.