Figure 2
An activated PERK pathway, during dengue infection, is essential for high autophagy turnover in MEF cells. (a) Bright-phase micrographs of PERK wild-type (PERK+/+) and PERK KO (PERK−/−) MEFs show adherent cells with minimal cytopathology. Dengue infection in PERK+/+ results in p62 degradation after 48 h, indicating a high autophagosome turnover, whereas treatment with ER stress inhibitor salubrinal (Sal) lowers turnover in infected PERK+/+ cells. We observed a more modest recovery of p62 after infection in PERK−/− cells as compared with the wild-type cells. Anti-dengue E protein (anti-E) signal is present in all infected cells; there is slightly less production in infected PERK−/− and Sal-treated PERK+/+ cells. (b) The graph indicating the total number of puncta per cell illustrates a significant decrease (P-value: 0.003) in the GFP puncta in infected PERK WT cells and recovered increase in puncta in infected cells exposed to Sal. (c) Western blot of whole-cell lysates, obtained after 24 h of mock and dengue (Den) infection, reveals an increase in the LC3 lipidation of PERK+/+ cells. PERK−/− cells, however, do not show a significant LC3 lipidation after 24 h of infection. (d) By Trypan blue exclusion assay, we observe a loss after 24 h of infection of dengue-conferred protection against CPT in PERK−/− MEFs compared with wild types. (e) Using plaque assay, we compared production of virus in PERK+/+ and PERK−/− cells after 48 h of infection. Dengue plaque formation decreases significantly (85%, 0.8 log10 units) in the PERK KO cells. The experiments were repeated at least three times
