Video S2 NA-deficient influenza virus does not spread in the absence of trypsin. Confluent MDCK cells were infected with the NA-deficient influenza virus at MOI of 0.0001 in the absence of trypsin. After incubation at 37uC for 24 hours, a single GFP-positive cell was detected, and then this cell and neighborhood cells was traced during the period from 24 hpi to 48 hpi at interval of 1 hour. Live cell imaging data analyses were performed by Biostation ID (GE healthcare). Scale bar, 50 mm. (MOV) Persistent Expression of Hepatitis C Virus Non-Structural Proteins Leads to Increased Autophagy and Mitochondrial Injury in Human Hepatoma Cells HCV infection is a major cause of chronic liver disease and liver cancer in the United States. To address the pathogenesis caused by HCV infection, recent studies have focused on the direct cytopathic effects of individual HCV proteins, with the objective of identifying their specific roles in the overall pathogenesis. However, this approach precludes examination of the possible interactions between different HCV proteins and organelles. To obtain a better understanding of the various cytopathic effects of and cellular responses to HCV proteins, we used human hepatoma cells constitutively replicating HCV RNA encoding either the full-length polyprotein or the non-structural proteins, or cells constitutively expressing the structural protein core, to model the state of persistent HCV infection and examined the combination of various HCV proteins in cellular pathogenesis. Increased reactive oxygen species (ROS) generation in the mitochondria, mitochondrial injury and degeneration, and increased lipid accumulation were common among all HCV protein-expressing cells regardless of whether they expressed the structural or non-structural proteins. Expression of the non-structural proteins also led to increased oxidative stress in the cytosol, membrane blebbing in the endoplasmic reticulum, and accumulation of autophagocytic vacuoles. Alterations of cellular redox state, on the other hand, significantly changed the level of autophagy, suggesting a direct link between oxidative stress and HCV-mediated activation of autophagy. With the wide-spread cytopathic effects, cells with the full-length HCV polyprotein showed a modest antioxidant response and exhibited a significant increase in population doubling time and a concomitant decrease in cyclin D1. In contrast, cells expressing the non-structural proteins were able to launch a vigorous antioxidant response with up-regulation of antioxidant enzymes. The population doubling time and cyclin D1 level were also comparable to that of control cells. Finally, the cytopathic effects of core protein appeared to focus on the mitochondria without remarkable disturbances in the cytosol. Hepatitis C virus (HCV) is an enveloped, positive, singlestranded RNA virus in the family of Flaviviridae [1] . The linear, non-segmented HCV genome of 9.6 kb encodes a polyprotein that undergoes post-translational cleavage by cellular and viral proteases to yield at least 10 mature proteins [2] [3] [4] . HCV infection is a major cause of chronic liver disease and is the major cause of liver cancer in the United States. HCV produces a chronic infection in 50-80% of infected patients; among them, roughly 20% will eventually develop liver cirrhosis. It is widely accepted that insufficient host immune response in eliminating HCV leads to persistent infection and the eventual development of liver diseases [4] [5] [6] . Interferon-a and ribavirin treatments have been prescribed either to stimulate immune response for clearance of viruses or to disrupt viral replication. However, high toxicity and low efficacy toward the two most prevalent HCV subtypes, 1a and 1b, in the US has been a barrier to effective eradication of persistent HCV infections [7] .