FIG 7.

ASFV pE66L inhibits host protein translation through the PKR/eIF2α signaling pathway. (A and B) PKR and eIF2α colocalize with pE66L. HEK-293T cells were transfected with the plasmid of E66L and analyzed at 24 h posttransfection by confocal microscopy using antibodies against HA (green), PKR (red) (A), or eIF2α (red) (B). Individual channels and merged images are shown. The white arrows indicate the cosubcellular localization between PKR or eIF2α and pE66L in the cells. Scale bars 5 μm. (C) The immunoprecipitated proteins (PKR and eIF2α) and pE66L were examined by Western blotting using anti-PKR, anti-eIF2α, and anti-HA antibody. The expression of GAPDH was detected with an anti-GAPDH monoclonal Ab to confirm equal protein loading. (D) HEK-293T cells were transfected with different doses of the E66L plasmid (0 to 2.0 μg) for 24 h. The cells were subjected to Western blot analysis (left) and immunoblotted with antibodies against phosphorylated PKR (p-PKR), PKR, phosphorylated eIF2α (p-eIF2α), eIF2α, and GAPDH. The grayscale values of the protein bands were analyzed by ImageJ (right). Data are presented as the means ± SD of three independent experiments; ns, not significant; *, P < 0.05; ***, P < 0.001. (E and F) HEK-293T (E) or IPI-2I (F) cell lysates were harvested at 24 h posttransfection, and the proteins were separated using Western blot analysis (left) and immunoblotted with antibodies against p-PKR, PKR, p-eIF2α, eIF2α, and GAPDH. The gray scale values of the protein bands were analyzed using ImageJ (right). Data are presented as the means ± SD of three independent experiments; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G and H) Expression of E66L and E66L(13–34) in the cells of panels (E) and (F). At 24 h posttransfection, the cells were fixed and incubated with a monoclonal antibody against the HA protein and then visualized by IF. Original magnification 200× (scale bars 100 μm).