Skip to main content
. 2016 Dec 23;5:e21198. doi: 10.7554/eLife.21198

Figure 4. HCV IRES binding alters natively purified PICs into complexes suited for IRES-driven translation.

(A, B, and D) Results of the experimental protocol shown in Figure 4—figure supplement 1. Natively purified 40S subunit-containing complexes were challenged with WT HCV IRES, then the composition of the complex (bound to 40S) and the factors released (released from 40S) were analyzed using western blot analysis. Panel (A): PICs from unstressed 293F cells. Panel (B): PICs from 293F cells stressed with dithiothreitol (DTT). Panel (D): PICs from unstressed cells incubated with non-hydrolyzable GTP analog GMP-PNP. (C) 80S ribosome formation on radiolabeled β-globin leader or HCV IRES in treated 293F lysate either untreated or treated with GMP-PNP. Reactions were fractionated by ultracentrifugation through a sucrose gradient. Total radioactivity per fraction is plotted. (E) Translation from bicistronic reporter mRNA (Figure 3A) containing WT or mutant HCV IRES, used to transfect Huh 7.5 cells that were left untreated (H2O control) or pretreated with DTT. Translation of Fluc is plotted relative to translation of WT HCV IRES reporter in unstressed cells (set to 100%). Error bars represent averages ±SEM of ≥3 independent experiments. Statistical significance shown by: *p<0.05, **p<0.01, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.21198.008

Figure 4—source data 1. Quantified blot data supporting Figure 4A–D.
Tables containing quantified band intensities from blots resulting from multiple biological replicates (n = 3). Blots were quantitated by densitometry, and the intensity of each eIF band was normalized to the rpS6 band. Graph shows the average of three independent experiments with the amount of each eIF bound to 40S subunit in the presence of WT HCV IRES set to 1. Errors represent one standard error from the mean. ND = not detectable.
DOI: http://dx.doi.org/10.7554/eLife.21198.009
elife-21198-fig4-data1.docx (13.1KB, docx)
DOI: 10.7554/eLife.21198.009

Figure 4.

Figure 4—figure supplement 1. Experimental protocol for analysis of HCV IRES remodeling of natively purified PICs.

Figure 4—figure supplement 1.

(A) Diagram of the PIC preparation strategy and experiment to analyze bound and released factors. (B) Western blot showing effective phosphorylation of eIF2α (p-2α) with both thapsigargin and DTT treatments. (C) Western blot analysis showing the presence of eIF2 in the three PIC preparations presented in Figure 4A,B and D. Inactive, phosphorylated eIF2α did not co-purify with these complexes. (D) Diagram of bicistronic reporter construct and translation activity in Huh 7.5 cells from two control IRESs: The eIF2-independent cricket paralysis virus (CrPV) intergenic region (IGR) IRES, and the eIF2-dependent encephalomyocarditis virus (EMCV) IRES. Treatment of the cells with thapsigargin induced eIF2α phosphorylation and reduced EMCV IRES-dependent translation, while the CrPV IGR IRES retained activity. For each construct, unstressed cap (black) and unstressed IRES (grey) activities are normalized to 100%. Error bars represent averages ±SEM of ≥3 independent experiments. (E) Translation initiation activity of the HCV IRES (compared to cap-driven translation) in Huh 7.5 cells, comparing stress induced by thapsigargin and DTT. Error bars represent averages ±SEM of ≥3 independent experiments. Statistical significance shown by: *p<0.05, **p<0.01, ***p<0.001. (F) Natively purified PICs from unstressed HeLa cells, analyzed for the effect of HCV IRES binding, as in Figure 4A.
DOI: http://dx.doi.org/10.7554/eLife.21198.010