MCV sT promotes 4E-BP1 S65 hyperphosphorylation and cap-dependent translation.
(A) MCV sT promoted δ 4E-BP1 hyperphosphorylation at S65; this phosphorylation was mediated by mTORC1 and could be inhibited by long-term raptor knockdown. 293 cells were stably transduced with Raptor shRNA lentivirus (shRap) and transfected with sT or empty vector on day 5 after transduction. (B) MCV sT prevented loss of hyperphosphorylated 4E-BP1 S65 during short-term mTORC1 inhibition with rapamycin. 293 cells transfected with empty vector or MCV sT were treated with 50 nM rapamycin for up to 1 hour. Basal phosphorylation at T37 and T46 was unaffected by rapamycin treatment or sT expression. (C) MCV sT prevented loss of 4E-BP1 during short-term mTORC1 and mTORC2 inhibition with Torin1 and PP242. 293 cells, with or without sT expression, were treated with Torin1 (500 nM) or PP242 (5 μM) for 6 hours. 4E-BP1 was almost completely dephosphorylated after drug treatment in the absence of sT expression. When sT was expressed, the δ S65 form was preserved. (D) Both wild-type sT and PP2A-binding sT mutants promoted rapamycin-resistant 4E-BP1 phosphorylation in 293 cells treated with 50 nM rapamycin for 1 hour.