During natural CHIKV infection, mTORC1 limits viral replication despite CHIKV requiring a host mRNA cap. ideals SEM from four self-employed experiments. Students test **, P 0.05.(PDF) ppat.1005091.s002.pdf (802K) GUID:?7293B49F-59F6-4108-A072-2DE3D69BF9E6 S3 Fig: The enhancement of mTORC1 activity the inhibition of TSC2 limits CHIKV infection. (AC) MEFs were pretreated with siRNA and Western blot was performed using anti-TSC2 and anti-GAPD antibodies. Additionally, cell lysates were assessed for tyrosine 389 phosphorylation of S6K-1 like a measure of mTORC1 activity (p-S6K1). Related results were observed in three self-employed experiments (A). MEFs were infected by CHIKV (MOI = 1) and 24h p.i., the percentage of E2 positive cells was measured. Bars show mean ideals SEM from three self-employed experiments (B). MEFs were infected by CHIKV-GFP (MOI = 1) and GFP positive cells were analyzed using real time imaging. Similar results were observed in five self-employed experiments (C). MEFs were infected by CHIKV (MOI = 1) in the presence of Rapamycin (100 nM) and 24h p.i. the percentage of E2 positive cells was identified. Bars show mean ideals SEM from three self-employed experiments (D). College students test **, P 0.05.(PDF) ppat.1005091.s003.pdf (1.1M) GUID:?E2E939C5-5120-4FB8-9847-23ECE43A2244 S4 Fig: Effect of mTORC1 on CHIKV infection is independent of type I IFN pathways. (A) Wild type (WT), or MEFs were infected with CHIKV (MOI = 0.01) for 24h and stained with an anti-E2 antibody. The percentage of E2 positive cells is definitely shown. Bars show mean ideals SEM from five self-employed experiments. (B) MEF cells were infected with CHIKV (MOI = 5) in the presence of TORISEL and Western Oligomycin blot was performed using anti-IB, anit-p-p65 and anti-GAPDH antibodies. Related results were observed in two self-employed experiments. (C) MEFs were infected with CHIKV at indicated MOI in the presence of TORISEL and the concentration of extracellular cytokines were analyzed at 24h post-infection. Bars indicate mean ideals SEM from three self-employed experiments. (D, E) MEFs were infected with CHIKV-GFP (MOI = 1) Oligomycin in presence of TORISEL and/or Actinomycin D (10 ng/ml). (D) GFP positive cells were analyzed during 50h of illness using real time imaging. Similar results were observed in three self-employed experiments. (E) Results represent the collapse induction of GFP positive cells observed in TORISEL treated cells as compared to untreated cells. Bars indicate mean ideals SEM from three self-employed experiments. Students test **, P 0.05.(PDF) ppat.1005091.s004.pdf (1.3M) GUID:?D61598AB-FDEB-42CE-90E9-EB1C86A2F6B0 S5 Fig: Inhibition of mTORC1 favors CHIKV infection independently of autophagy. (A-D) or were infected with CHIKV (MOI = 1) for 24h in presence of Rapamycin or TORISEL. The manifestation of Atg5, Atg7 and GAPDH was monitored (E); the percentage of E2 positive cells was determined by FACS analysis (F); and extracellular viral titers were analyzed (G). Bars show mean ideals SEM from three self-employed experiments. +, shows respective siRNA knock-down;-, indicates si-control. College students test **, P 0.05(PDF) ppat.1005091.s005.pdf (1.0M) GUID:?9C6A5C58-707E-4042-9CC3-0B96D690998B S6 Fig: TORISEL decreases mTORC1 activity. (A, B) mice were treated with intra-peritoneal injection of 100 L of remedy containing TORISEL (10 mg/kg) or PBS for 8 days. Pores and skin (A) and muscle mass (B) were collected and mTORC1 activity was assessed by Oligomycin following tyrosine 389 phosphorylation of S6K-1 (p-S6K1) by Western blot. Results are representative of three self-employed experiments. ?, control; T, TORISEL.(PDF) ppat.1005091.s006.pdf (238K) GUID:?DBAA1A87-F66D-43D5-92B8-9A529D7E172D S7 Fig: CHIKV infection requires eIF4E activity to replicate. (Abdominal) MEFs were pre-treated with siRNA followed by CHIKV illness (MOI = 1). Western blot using anit-eIF4E and anti-GAPDH was performed and the percentage of E2 positive cells was analyzed at 24hrs p.i. Bars show mean ideals SEM from Robo3 three self-employed experiments (A). MEFs were infected with CHIKV-GFP (MOI = 5) and GFP positive cells were analyzed using real time imaging. Similar results were observed in four self-employed experiments (B). (C) MEFs were infected with CHIKV-GFP (MOI = 5) in presence of 4EGI-1 (4EGI, 10 M) and GFP positive cells were analyzed using a real time imaging system. Related results were observed in five self-employed experiments. (D) MEFs were infected with an increasing dose of the CHIKV-Luc recombinant in presence of 4EGI-1 and the luciferase activity was measured 4h p.i. Bars show mean ideals SEM from three self-employed experiments. Students Oligomycin test **, P 0.05.(PDF) ppat.1005091.s007.pdf (620K) GUID:?D5A516AC-0F54-4604-9F36-5106820A8A3C S8 Fig: CHIKV-mediated inhibition of mTOR provides a counter-balance for the antiviral effect of ROS production. (A, B) MEF were infected with CHIKV at indicated MOI in the presence of a ROS inhibitor (siRNA followed by CHIKV illness at indicated MOI. The percentage of intracellular E2 staining was analyzed as described inside a. Error pub indicate imply ideals SEM from four self-employed experiments. Students test **, P 0.05.(PDF) ppat.1005091.s008.pdf (677K) GUID:?FA4C34F4-D37E-41BE-844F-731FD266C873 S1 Movie: (Related to Fig 4) mTORC1 inhibitors enhance translation of both structural and.