Figure 5.

Analysis of NSs-induced PKR downregulation. The Fig is adapted from Ikegami et al.^[26] (A) 293 cells were mock infected (Mock) or infected with MP-12 (MP-12) at an moi of 3. Whole-cell lysates were collected at 2, 4, 6 and 8 h.p.i. Anti-PKR antibody, anti-NSs antibody and anti-β-actin antibody were used to detect PKR, NSs and β-actin, respectively. (B) 293 cells were mock infected (Mock) or infected with MP-12 at an moi of 3 or transfected with in vitro-synthesized RNA transcripts encoding NSs. Cells were then mock-treated or treated with 100μg/ml of puromycin. Cell extracts were harvested at 16 h.p.i. or 16 h post transfection, and the abundance of PKR and viral proteins were analyzed by Western blotting with anti-PKR antibody (top panel), anti-RVFV antibody (middle panel) or anti-β-actin antibody (bottom panel). (C) 293 cells were mock-transfected or transfected with in vitro-synthesized RNA transcripts encoding MP-12 NSs or rLuc. Cells were mock-treated or treated with 5 μg/ml of ActD. Total RNA was harvested at 8 h post transfection, and analyzed by real-time PCR. The relative abundance of PKR mRNA of each sample was calculated by the ΔΔC_T method based on the abundance of 18S ribosomal RNA. The data shown in the graph (mean +/- standard deviation) were obtained from three independent experiments. The p value was determined by Student's t-test (*: p<0.05). (D) 293 cells were infected with rMP12-rLuc or MP-12 at an moi of 3, and, then, treated with 10 μM of MG132 (MG) or 50 μM of lactacystin (LA) or they were mock treated (-). Whole-cell lysates were collected at 8 h.p.i. and the abundance of PKR, NSs and β-actin was examined by Western blot analysis.