Uncoupling the effects of rocaglates on translation initiation versus NMD. (A) Reporter constructs used in these assays. In constructs FF-4.04 and FF-4.06, the FLuc cistron is fused to β-globin exons lacking (FF-4.04) or harboring (FF-4.06) a PTC (denoted by an asterisk). (B) Luciferase activity following introduction of FF-4.04 + HCV/RLuc or FF-4.06 + HCV/RLuc into 4A1F163L/4A2− Hap1 cells. Following transfections, cells were incubated for 1 d, reseeded, and exposed to compound for 6 h at which point luciferase activity was determined. n = 3 ± SD. (C) Constructs HCV-4.04 and HCV-4.06 have the HCV IRES engineered upstream of the FLuc coding region. Red asterisk indicates the location of the PTC. (D) Luciferase activity following introduction of HCV/FF-4.04 + HCV/RLuc or HCV/FF-4.06 + HCV/RLuc into 4A1F163L/4A2− Hap1 cells. Following transfections, cells were incubated for 1 d, reseeded, and exposed to compound for 6 h at which point luciferase activity was determined. n = 3 ± SD. (E) RNA levels in CR-1-31B and CHX treated FF-4.04 and FF-4.06 cells. 4A1F163L/4A2− eHAP1 cells were nucleofected with FF-4.04 or FF-4.06 and selected as a pool in G418 (1.6 mg/mL). Cells were exposed to CR-1-31B or CHX for 5 h, after which time RNA was isolated and analyzed by RT-qPCR. RNA levels were normalized to GAPDH levels and expressed relative to DMSO controls. n = 4 ± SD. (***) P = 0.0005; ns, not significant.
