Figure 1.

QL47 inhibits protein synthesis in live cells. A, chemical structures of QL47 and the derivatives YKL-04-085, QL47R, and YKL-03-109 (compound 14) (6, 9). QL47R is an inactive analog due to replacement of the cysteine-reactive acrylamide moiety with a nonreactive propyl amide. Compound 14 is a negative control compound with significantly diminished antiviral activity. B, HEK293T cells were transfected with an in vitro transcribed reporter DV subgenomic RNA bearing the virus's seven nonstructural genes (NS1–NS5) as well as a NanoLuc®-proline, glutamate, serine, threonine (NlucP) luciferase reporter gene. The intracellular reporter activity was immediately measured for 1 h at 37 °C. Cells were treated with DMSO or 2 μm QL47 at 1 h post-transfection, and the intracellular luciferase signal was then measured continuously for 3 h at 37 °C. The luciferase signal obtained from mock-transfected cells was subtracted, and data are presented as means ± S.D. of three experimental replicates. One representative experiment is shown from two independent experiments. RLU, relative light units. C, measurement of puromycin incorporation in nascent cellular proteins. Huh7 cells were treated with DMSO, 50 μg/ml CHX, or 2 μm QL47 for 1 h. The cells were next pulse-labeled with 1 μm puromycin for 30 min, and their cellular contents were analyzed by Western blotting. Prematurely terminated polypeptides were detected using a puromycin-specific antibody. Their abundance was normalized to the loading control (tubulin) and is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from two independent experiments. D, metabolic labeling of cellular proteins with radiolabeled amino acids. Huh7 cells were starved for 30 min in methionine/cysteine-free medium and concomitantly treated with DMSO, 30 μg/ml CHX, 2 μm QL47, or 2 μm compound 14. The medium was next supplemented with a mixture of ³⁵S-Met and ³⁵S-Cys for 30 min. Total cell lysates were analyzed by SDS-PAGE, followed by autoradiography to measure bulk protein synthesis. Neosynthesized protein abundance is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from three independent experiments. E, Huh7 cells were treated with DMSO or 2 μm QL47, and metabolic labeling (red boxes) was performed as indicated in D 1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in D, and their abundance is presented as a percentage of the DMSO-treated control samples at each time point. One representative experiment is shown from two independent experiments.