Fig 6. Inhibition of mTORC1 favors viral protein translation by activation of an MnK/p-eIF4E pathway.

(A) MEFs were infected with CHIKV (MOI = 5) in the presence of TORISEL (0.1 mg/ml) for 24h and eIF4E phosphorylation was assessed by Western blot. Band intensity of the p-eIF4E/GAPDH ratio calculated with numbers shown below the respective conditions. Similar results were observed in three independent experiments. (B) MEFs were pretreated with eif4e siRNA followed by CHIKV infection (MOI = 1) in presence of Rapamycin (10 nM) or TORISEL (0.1 mg/ml). Percentage of E2 positive cells was measured 24 p.i.. Bars indicate mean values ±SEM from three independent experiments. (C) MEFs were infected with CHIKV (MOI = 5) in presence of TORISEL (T) and/or an MnK1/2 inhibitor (CGP57380, 20 μM) for 24h and Western blot analysis was performed to detect phosphorylation of eIF4E at serine 209 (p-eIF4E (S209)). eIF4E and GAPDH were measured to control protein expression and loading. Band intensity of the p-eIF4E / GAPDH ratio is reported. (D) MEFs were infected with CHIKV-GFP (MOI = 1) in the presence of TORISEL and/or indicated dose of an MnK1/2 inhibitor (CGP57380). GFP positive cells were analyzed 24 p.i. using real time imaging. Results represent the fold induction of GFP positive cells observed in TORISEL treated cells, as compared to untreated cells. Bars indicate mean values ±SEM from three independent experiments. (E) MEFs were infected with increasing doses of CHIKV-Luc in presence of TORISEL ± MNKs inhibitor II (MNK1/2 inhi. – 5 μM) and luciferase activity was measured at 4h p.i. Bars indicate mean values ±SEM from three independent experiments. (F) MEFs were infected with CHIKV (MOI = 5) for 24h in presence of TORISEL ± MEKs inhibitor (PD0325901, 1 μg/ml), an p38 MAPK inhibitor (SB203580, 1 μM) or an PI3K inhibitor (LY294002, 25 μM) and eIF4E phosphorylation was followed by Western blot. p-eIF4E / GAPDH band intensity is reported. Similar results were observed in three independent experiments. (G) MEFs were infected with CHIKV (MOI = 1) for 24h using the inhibitors described in (E). Results represent the fold induction of E2 positive cells observed in TORISEL treated cells, as compared to untreated cells. Bar indicate mean values ±SEM from three independent experiments. (H, I) MEFs were pretreated with siRNA specific for mtor, s6k1 or 4e-bp1 followed by CHIKV infection (MOI = 1) in presence of TORISEL. Western blot was performed 24h p.i. using anti-mTOR, anti-S6K1 and anti-4E-BP1 antibodies (H). Additionally, the fold induction of E2 positive cells is shown. Bars indicate mean values ±SEM from three independent experiments (I). Ø means control; T means TORISEL. +, indicates respective siRNA knock-down;-, indicates si-control. Student’s test **, P < 0.05.