Figure 10. The L uAUG modulates translation of the L pORF in response to eIF2α phosphorylation in 293T cells.

A. Diagram of the pCAGGS EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF) and an identical construct, lacking the uAUG codon (Lns-FF). B. Thapsigargin (TG) treatment induces eIF2α∼P. Cells were treated with either DMSO or with increasing doses of TG and lysed in NP-40 lysis buffer with protease inhibitors. eIF2α∼P levels were measured by western blot. C. Western blot of total eIF2α and eIF2α∼P levels, from untreated and TG-treated cells which were lysed in passive lysis buffer that was used for the luciferase analysis in panel D. D. The L uAUG functions to maintain L translation following TG treatment. 293T cells were transfected with pRLTK and the L-FF or the Lns-FF reporter constructs. At 24 hpt, cells were treated with four doses of TG and harvested at 10 hours post treatment. Dual luciferase assays were performed to determine the firefly to Renilla luciferase ratio in the presence or absence of TG treatment and the FF to Renilla ratio of the DMSO treated cells transfected with L-FF was set to 1. Each data point represents the mean and standard deviation of four replicates.