Figure 5. PKR is required to control CHIKV infection and IFN-β production in MEFs.

A) WT and PKR^−/− MEFs were infected with CHIKV-GFP at an MOI of 10 or 50, for 24 h and 48 h. The amount of infected cells was determined by GFP expression, left panel. Interferon β present in the cell culture supernatants was measured by ELISA, right panel. Data represented are mean ± standard deviation from 3 experiments. B) WT (top panel) and PKR^−/− MEFs (bottom panel) were infected for 24 h with CHIKV-GFP, then labeled with puromycin for 1 h prior fixation. GFP-CHIKV positive (green) were visualized by confocal microscopy after staining with specific antibodies for puromycin (cyan) and phospho-eIF2α (red). Cell Nuclei are stained with Hoechst 33258 (blue). Infection by CHIKV inhibits protein synthesis (visualized by puromycin incorporation) in WT, but not in PKR-deficient cells (arrows). In WT MEFs, eIF2α phosphorylation levels correlate with translation inhibition, although variability is observed among different infected cells, presumably due to GADD34 activity and time of infection. Non-infected WT and PKR^−/− cells serve as a reference for normal translation activity and are indicated by an arrowhead. Scale bar 10 µm.