mTORC1 inhibitors enhance translation of both structural and non-structural CHIKV proteins.

<p><b>(A)</b> MEFs were infected with increasing doses of CHIKV-luciferase reporter construct in the presence of TORISEL. A schematic of the CHIKV-luciferase construct is depicted. Luciferase activity was measured 4h p.i. using luciferin and read-out on a luminometer. Bars indicate mean values ±SEM from three independent experiments. <b>(B)</b> A schematic of the CHIKV-GFP construct is illustrated. MEFs were infected with CHIK-GFP (MOI = 1) in the presence of Rapamycin (100 nM) or TORISEL (0.1 mg/ml). GFP positive cells were quantified using real time imaging. Images show GFP staining (used to define green object count) and curves plot the kinetics of infection. Similar results were obtained in seven independent experiments. <b>(C)</b> MEFs were infected with CHIKV-GFP for 24h at indicated MOI in presence of Rapamycin or TORISEL, and the percentage of GFP positive cells were determined. Similar results were observed in five independent experiments. <b>(D)</b> WT MEFs were infected with CHIKV-GFP (MOI = 1) for 24h in presence of TORISEL. Infected cells were gated according to GFP intensity and the MFI was plotted for infected cell populations. Bar indicate mean values ±SEM from three independent experiments. <b>(E)</b> A schematic of the single cycle rCHIKV-GFP construct is shown. MEFs were transfected with rCHIKV-GFP and after 24h TORISEL was added to the cultures. GFP positive cells were gated by flow cytometry and the MFI was plotted. Bars indicate mean values ±SEM from three independent experiments. <b>(F)</b> Cell lysates were prepared from rCHIKV-GFP transfected MEFs and Western blot was performed using anti-GFP and anti-GAPD antibodies. Similar results were observed in two independent experiments. Ø, control; T, TORISEL. Student’s test **, P < 0.05.