mTOR inhibitors lower an intrinsic barrier to virus infection mediated by IFITM3

Shi, Guoli; Ozog, Stosh; Torbett, Bruce E.; Compton, Alex A.

HeLa cells were treated with DMSO or rapamycin (20 uM) for four hours and total RNA was extracted with Tri-Reagent (Sigma). RNA was incubated with Turbo DNAse I (Ambio) for one hour at 37°C followed by first-strand cDNA synthesis using the SuperScript III kit (Invitrogen). Quantitative PCR of cDNA templates was run on a DNA Engine Opticon 2 detection system using Power SyberGreen PCR Master Mix (Applied Biosciences) paired with the following primer sets: 5'-GAATCACACTGTCCAAACCTTC-3' and 5'-CATAGGCCTGGAAGATCAGC-3' (IFITM3); 5'-GAAATCCCATCACCATCTTCCAGG-3' and 5'-GAGCCCCAGCCTTCTCCATG-3' (GAPDH). HeLa cells were seeded in 24-well plates (50,000/well) overnight and treated with DMSO or the indicated concentration of Torin 1 for four hours. Cells were fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. (C) HEK293T cell lines stably expressing FLAGtagged IFITM3, IFITM3 mutants, IFITM2, or IFITM1 were seeded in 24-well plates (50,000/well) overnight and treated with DMSO or rapamycin (30 uM) for four hours. Cells were fixed/permeabilized, stained with anti-IFITM3, and analyzed by flow cytometry. MFI from univariate histograms of IFITM3 or FLAG expression were normalized and averaged, with expression in the DMSO condition of each cell line set to 100. Error bars indicate standard error from three to five experiments.