To confirm the initial observation, immunocytochemical and western blot analyses were carried out to determine the status of LC3, which is an integral part of the autophagosome membrane. LC3-positive punctuate structures in the cytoplasm were prominent in genome-length replicon and subgenomic replicon cells ( Figure 3C ), but they were only present at a very low level in Coreon cells. Consistent with this result, western blot analyses showed a marked increase of LC3-I and LC3-II in genome-length replicon and subgenomic replicon cells ( Figure 3D ). However, the ratios between LC3-I and II were not changed. In contrast to the prominent accumulation of autophagocytic vacuoles in genomelength and subgenomic replicon cells, autophagosomes were not detected in HCV transgenic mice expressing the full-length HCV polyprotein (data not shown).