FIG 6.

The activated PERK-eIF2α pathway of the UPR inhibits TGEV replication. (A and B) Inhibition of PERK by GSK2606414 promoted TGEV replication in ST cells as measured by Western blotting using antibodies against the TGEV N protein (A) and viral titers (B). (C) ST cells were transfected with shRNA targeting PERK or control shRNA for 24 h, and then the cells were challenged with TGEV. At 24 hpi, Western blot analyses for PERK, eIF2α, p-eIF2α, TGEV N, or β-actin were performed. (D) ST cells were transfected with PERK shRNA number 2 or control shRNA for 24 h and then challenged with TGEV for 6, 12, 24, 36, and 48 h. Viral titers were calculated at different time points. (E and F) ST cells were treated with the indicated concentrations of salubrinal for 24 h and then infected with TGEV H87 (MOI = 1) in the continued presence of salubrinal. (E) After the virus adsorbed to the cells, medium containing the indicated concentrations of salubrinal was added. Cell lysates of ST cells were analyzed by Western blotting using antibody against p-eIF2α and the TGEV N protein. (F) The TGEV TCID₅₀ in the supernatants was titrated on ST cells. (G and H) ST cells were transfected with HA-eIF2αwt or HA-eIF2αS51A for 24 h and then infected with TGEV H87. At 24 hpi, Western blot analysis was performed to detect p-eIF2α, TGEV N, or β-actin, and the viral titers were calculated. (I and J) ST cells were transfected with siRNA of eIF2α for 24 h and then infected with TGEV H87. After 24 hpi, Western blot analysis was performed to detect eIF2α, TGEV N, or β-actin, and the viral titers were calculated. Means and SD of the results from three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.