FIGURE 9.

Flavivirus NS4A-induced protection in epithelial cells is dependent upon autophagy. A, Western blotting using a specific antibody for cleaved LC3II reveals an increase in LC3II cleavage following both live Dengue-2 and Modoc infections, as well as after individual expression of NS4A from either virus. B, β-tubulin was used as a loading control. C, whole-protein lysates were collected from MDCK cells transiently expressing Dengue-2 nonstructural proteins NS1, NS2A, NS4A, and NS4B at 24 h post-transfection, as well as cells transfected with empty plasmid and cells exposed to the transfection agent as controls. All conditions stimulate a moderate amount of LC3 cleavage, likely because of stress occurring during our transfection protocol as indicated by LC3 cleavage after exposure to the transfection agent alone. NS4A expression leads to a substantial increase in LC3 cleavage compared with controls and expression of other Dengue-2 nonstructural genes, indicating NS4A expression alone is uniquely sufficient to up-regulate autophagy in MDCK cells. D, β-tubulin was used as a loading control. E, mock − infected HeLa cells stably expressing LC3-GFP exhibit little LC3-GFP punctation, whereas Dengue-2 (F) and Modoc (G) virus infections induce LC3-GFP punctation following infection, indicating autophagy up-regulation. H, expression of empty plasmid vector control in MDCK cells transiently expressing LC3-GFP does not induce LC3-GFP punctation, whereas expression of Dengue-2 NS4A (I) or Modoc NS4A (J) both induce LC3-GFP punctation, indicating autophagy up-regulation following NS4A expression.