Figure 1. Analysis of HCV IRES preinitiation complex composition, formed in lysate.
(A) Results of pull-down experiments. HCV IRES was 5’ biotinylated and immobilized on ’ streptavidin agarose beads. Factors and subunits that bound to the IRES after incubation in lysate were detected by western blot analysis. Lane 1: Input lysate. Lane 2: streptavidin beads only. Lane 3: CrPV IGR IRES RNA control. Lane 4: WT HCV IRES RNA. Lane 5: HCV IRES mutant dIIId_GGG-CCC. (B) Secondary structure cartoon of the HCV IRES RNA, with the location of mutations indicated. (C) Translation initiation activity of the HCV IRES, WT and mutants, with RNA transfections of Huh 7.5 cells. Error bars represent averages ±SEM of ≥3 independent experiments. (D) Pull-downs as in panel (A) with HCV IRES mutants shown in panel (B). 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 WT set to 1. Error bars represent one standard error from the mean. (E) Pull-downs as in panel (A) with truncated HCV IRESs. Cartoon diagrams of these three truncations are shown, as is the portion of the IRES in which these truncations were made. Visual examination shows a similar qualitative pattern. (F) Pull-downs as in panel (A) with the IRESs from CSFV and SPV-1, along with corresponding secondary structure cartoons.
Figure 1—figure supplement 1. HCV IRES structure and clash between HCV IRES domain II and eIF2.
