Fig 3. Rapalog treatment enhances viral replication.

(A) MEFs were pretreated with TORISEL (0.1 mg/ml) then infected with CHIKV for 1h at 4°C. Extracellular E2 staining was analyzed by flow cytometry. Representative E2 staining is shown by histogram. Similar results were obtained in four independent experiments. (B) MEFs were pretreated with Rapamycin (100 nM) or TORISEL (0.1 mg/ml) for 24h and then infected with CHIKV (MOI = 2) for 2h. Intracellular E2 staining was analyzed with similar results obtained in four independent experiments. (C) MEFs were infected with CHIKV using indicated MOI in presence or absence of TORISEL (0.1 mg/ml). Northern blots were performed using specific radioactive probes that recognize 49S genomic or 26S subgenomic mRNA of CHIKV. Similar results were observed in three independent experiments. (D-H) MEFs were infected with CHIKV for 24h with indicated MOI in presence of Rapamycin (100 nM) or TORISEL (0.1 mg/ml). Blue bars indicate the period of Rapalog exposure. At the end of Rapalog treatment, cells were washed and maintained in control media or analyzed using flow cytometer depending of the time point (D). (E-H) E2 positive cells were represented in graphs corresponding to the time points of Rapalog exposure: 1h pretreatment, prior to infection (E); treated 2h during CHIKV infection phase (F); treated post-infection for 22h (G); or treated 24h p.i. for an additional 24h (G). Bars indicate mean values ±SEM from three independent experiments. Student’s test **, P < 0.05. NS, non significant.