FIG 7.

Analysis of mRNA translation by OPP labeling. (A and B) ST cells were pretreated with Tg (1 μM) or a combination with 3MA (5 mM) 2 h before infection and maintained at that concentration after infection. TGEV titers were calculated (A), and Western blotting was performed to test LC3 and TGEV-N expression, and β-actin was used as sample loading control (B). (C) TGEV infection decreased global protein synthesis. ST cells were infected with TGEV H87 (MOI = 1) or mock infected for 6, 12, 24, 36, or 48 h before OPP labeling. The graph shows the fold increase in OPP labeling (means and SD) in ST cells, with values for mock-infected cells set to 1.0. Statistical comparisons between control and TGEV-infected cells are shown (Student's t test). Cells treated with Tg (1 μM) were used as a positive control. (D, E, and F) ST cells were treated or not with 200 nM ISRIB or Tg (1 μM) for 2 h, followed by infection with TGEV H87 for 24 h before OPP labeling. (D) Graph showing fold increase in OPP labeling, with values for untreated cells set to 1.0. Statistical comparisons between groups are shown (Student's t test). (E) Immunoblot analysis of ATF4 and TGEV-N in TGEV-infected ST cells in the presence or absence of 200 nM ISRIB. (F) Virus titers were measured in ISRIB-treated cells. (G) ST cells were transfected with HA-eIF2αwt or HA-eIF2αS51A and analyzed for ongoing translation. (H) ST cells were transfected with eIF2α siRNA or control siRNA for 24 h and then infected with TGEV H87. At 24 hpi, global protein synthesis was measured by OPP labeling. Means and SD of the results from three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.