Figure 4

eIF2α is critical for NDV replication. A. HeLa cells were transfected with the siRNA specific for eIF2α (100 pmol/mL) or control siRNA for 48 h and then processed as described for Figure 3A. B. The intensity band ratio of eIF2α to β-actin. Representative results are shown with graphs representing the ratio of eIF2α to β-actin normalized to the control condition. Data are presented as means from three independent experiments. Significance was analyzed using two-tailed. Student’s t-test. ***, p ≤ 0.001. C. Determination of NDV replication in eIF2α knockdown HeLa cells. The extracellular virus yields were determined at 12 hpi and expressed as TCID₅₀/mL. Data are presented as means from three independent experiments. Significance was analyzed using two-tailed Student’s t-test. *, p ≤ 0.05. D. HeLa cells were infected with ZJ1-GFP at an MOI of 1 or mock infected for 9 h in the presence of OA (10 nM). The cells were harvested and then processed as described for Figure 1A. E and F. The intensity band ratio of peIF2α (E) or pPKR (F) to β-actin. Representative results are shown with graphs representing the ratio of peIF2α (E) or pPKR (F) to β-actin normalized to the control condition. Data are presented as means from three independent experiments. Significance was analyzed using two-tailed Student’s t-test. ***, p ≤ 0.001. G. HeLa cells were infected with ZJ1-GFP at an MOI of 1 for 9 h in the presence of OA (10 nM) and observed by immunofluorescenct microscopy. The number of GFP-positive cells was quantified using Image J software. Data are presented as means from three independent experiments. Significance was analyzed using One-way ANOVA followed by Tukey’s test. **, p ≤ 0.01; ***, p ≤ 0.001, *, p ≤ 0.05.