Figure 6

CHIKV nsP4 suppresses the phosphorylation of eIF2α. A) Schematic diagram showing the organization of CHIKV genes in the viral genome. CHIKV genes (nsP1-4, C, E1 &E2) were cloned in a CMV driven GFP tagged vector and restriction analysis in Figure 6A shows expected size insert in respective positive clones. B) HEK293 cells were transfected with 1 μg each of the recombinant CHIKV-gene plasmids and incubated for 24 h to permit protein translation. At 24 h, Western blotting was performed with anti-GFP antibody for the detection of the correct size proteins. Cells treated with transfection reagent without plasmid were used as negative control (CC). C) HEK293 cells were transfected with 1μg each of CHIKV-gene carrying plasmids or control GFP-gene plasmid for 24h and then treated with tunicamycin (0.5 μg/ml) for 24 h followed by immunofluorescence detection using antibody against phospho (Ser 51) eIF2α (red) under the confocal microscope and images were captured at 63X magnification. Nuclear stain DAPI (blue) was used to differentiate single cells and individually expressed CHIKV proteins were visualized as GFP-fused proteins (green). As indicated using red arrows staining of phospho (Ser 51) eIF2α (red) was dramatically reduced in cells expressing CHIKV nsP4. Images were processed using Zen image software from Zeiss. D) Cells (HEK293) were transfected with C, nsP2 and nsP4 or a control plasmid GFP for 24 h and then further treated with tunicamycin (0.5μg/ml) for 24 h followed by Western blotting using antibodies against phospho (Ser 51) eIF2α, and eIF2α. Anti-GAPDH was used as loading control and anti-GFP was used to probe plasmid encoded protein expressions. Change in band intensity of phospho (Ser 51) eIF2α in GFP and nsP4 transfected cells was calculated using image-J and presented as % eIF2α-P over total eIF2α. The data is representative of two independent experiments. The symbol (*) denotes p < 0.05.