80S assembly and structural probing of the chimeric IGR IRESs. (A) 80S-IGR IRES binding was monitored by competition titration experiments. The fraction of radiolabeled wild-type CrPV IGR IRES bound to 80S ribosomes is plotted against the log of the indicated unlabeled IRES RNA. Representative curves and quantitations are shown from at least three independent experiments. (B,C) Enzymatic probing of the chimeric IGR IRESs. Dicistronic RNAs containing the wild-type and chimeric IGR IRESs were treated with RNase T1 as indicated. Primer extension was performed as described in Materials and Methods using PrEJ69. The reaction products were separated on a denaturing polyacrylamide gel. The nucleotides that are cleaved 3′ by RNase T1 are indicated to the right. A sequencing ladder of the dicistronic construct using the appropriate primer is shown on the left.

Ribosome positioning on the chimeric IGR IRESs by toeprinting analysis. Dicistronic RNAs containing wild-type or mutant IGR IRES were incubated with (A) 40S alone or 40S and 60S subunits (100 nM), or (B) in RRL in the presence of edeine as described in the Materials and Methods. Reactions were analyzed by primer extension analysis using oligo PrEJ69. Reaction products were separated by denaturing polyacrylamide gels. The gels were dried and exposed by autoradiography. The location of major toeprint A and toeprint B, are shown on the right. Sequencing ladders for the wild-type IRESs are shown on the left, with their respective nucleotide numbers as indicated. The intensity of toeprint A in B is shown as a percentage of the total radioactivty in each lane, normalized to the wild-type CrPV IGR IRES (100%).

Ribosome-dependent GTPase stimulation of eEF2 by the chimeric IGR IRES. GTP hydrolysis was monitored by incubation of 80S, eEF2, and the indicated IRES with radiolabeled [γ-³²P]GTP. Aliquots of the reaction were quenched over time and then resolved by thin-layer chromatography. The slope of the linear regression lines, which represents the mean percent hydrolysis of radiolabeled GTP per minute, is shown in a bar graph. These values were derived from three independent experiments, and the error bars correspond to a 95% confidence interval. Note that ribosomes alone (-IRES/-eEF2) do not stimulate GTPase activity.

The secondary structure of the (A) Type I CrPV IGR IRES and the (B) Type II TSV IGR IRES. Conserved nucleotide positions are shown in uppercase and nonconserved nucleotides are in lowercase. Numbering refers to the nucleotide position within the respective viral genome. Helical regions are indicated by a black dash between nucleotides. Underlined nucleotides represent the first two amino acid residues in the viral capsid protein. Properly positioned 40S and 80S ribosomes on the IGR IRES produces a toeprint, denoted Toeprint A, shown by the arrows. The point at which the two classes of IGR IRES were swapped is indicated by a black line. The ΔSLIII deletion within the Type II TSV IGR IRES is marked by a blue box. L1.1A and L1.1B are indicated by red and blue letters, respectively.

Translational activities of the chimeric IGR IRESs. (A) Uncapped dicistronic RNAs containing wild-type or chimeric IGR IRESs were incubated in RRL at 30°C for 60 min in the presence of [³⁵S]methionine. The first cistron, encoding Renilla luciferase (Rluc), measures scanning-mediated translation, and the second cistron, firefly luciferase (Fluc), measures IGR IRES-mediated translation. Shown is a representative gel of radiolabeled Fluc and Rluc protein products detected by autoradiography. Where applicable, the amount of the ternary complex inhibitor NSC119889 added to the reactions is shown above the gel. (B) Quantitations of translational activities of the chimeric IRESs. The ratios of firefly to Renilla luciferase are shown and are normalized to the ratio of the dicistronic RNA containing the wild-type CrPV IGR IRES. (C) Normalized quantitation of the chimeric IRES translation under NSC119889 treatment. Firefly (top) and Renilla (bottom) luciferase activities were normalized to the translational activity of each dicistronic RNA in the absence of NSC119889. The data shown are the averages of at least three independent experiments ±SD. (D) Translational activities of the chimeric IRESs in the presence of edeine. Shown is a representative gel of radiolabeled Fluc and Rluc detected by autoradiography. The bottom panel shows quantitations of Rluc and Fluc, normalized to the amount of luciferase produced by each dicistronic RNA in the absence of edeine. The average value from three independent experiments ±SD is shown.

The activity of L1.1 IGR IRES chimeras. (A) Dicistronic RNAs containing wild-type or chimeric IGR IRESs were incubated in RRL at 30°C for 60 min in the presence of [³⁵S]methionine. The first cistron, encoding Renilla luciferase (Rluc), measures cap-mediated translation, and the second cistron, firefly luciferase (Fluc), measures IGR IRES-mediated translation. Shown is a representative gel of radiolabeled Fluc and Rluc protein products detected by autoradiography, and the IRES mutants tested are labeled at the top of the gel. (B) Amounts of Fluc and Rluc made are quantitated below the gel by PhosphorImager analysis, shown as a ratio between Fluc and Rluc. Results were normalized against wild-type CrPV IGR IRES. (C) The fraction of radiolabeled wild-type CrPV IGR IRES bound to 80S ribosomes is plotted against the log of the indicated unlabeled IRES concentration. Representative quantifications are shown from at least three independent experiments.

Characterization of SLIII within the TSV IGR IRES. (A) Dicistronic RNAs containing wild-type or ΔSLIII TSV IGR IRESs were incubated in RRL at 30°C for 60 min in the presence of [³⁵S]methionine. The first cistron, encoding Renilla luciferase (Rluc), measures scanning-mediated translation, and the second cistron, firefly luciferase (Fluc), measures IGR IRES-mediated translation. Shown are radiolabeled firefly (Fluc) and Renilla (Rluc) luciferase protein products detected by autoradiography and quantitated by PhosphorImager analysis. (B) 80S assembly on TSV and ΔSLIII IGR IRESs was assessed in RRL by sucrose gradient analysis. Radiolabeled wild-type or ΔSLIII IGR IRES (100 nM) was incubated in RRL with 0.1 mg/mL cycloheximide in the presence (+NSC) or absence (−NSC) of 25 μM NSC119889 for 15 min at room temperature. Reactions were loaded on a 10%–30% sucrose gradient and shown are the percent of total radioactive counts in each fraction. The top and bottom of the gradient is represented from left to right, respectively. Fractions containing free IRES, 40S, and 80S ribosomes are indicated. (C) Toeprint analysis of assembled 40S and 80S ribosomes on TSV and ΔSLIII IGR IRESs. 40S alone or 40S and 60S subunits (100 nM) were incubated with dicistronic RNAs containing wild-type or mutant IGR IRES and analyzed by primer extension analysis using oligo PrEJ69. Reaction products were separated in denaturing polyacrylamide gels. The gels were dried and exposed by autoradiography.