Figure 4.

Ribosome recruitment to the HCV IRES is refractory to NSC119889. Krebs-2 extracts were preincubated with 50 μM NSC119889 and either 1 mM GMP-PNP (A and B) or 0.6 mM cycloheximide (C and D) at 30°C for 5 min. These were supplemented with ³²P-radiolabeled CAT, HCV IRES, or CrPV IRES RNA, and then incubated for an additional 10 min at 30°C. Initiation complexes were resolved by centrifugation through the gradients indicated below the graphs to resolve different complexes. The percentage of complexes formed were (A) CAT mRNA/GMP-PNP (48S, 11.9%); CAT mRNA/GMP-PNP + NSC119889 (48S, <0.6%) and (B) HCV IRES/GMP-PNP (40S, 10.2%; and 48S, 63.5%), HCV IRES/GMP-PNP + NSC119889 (40S, 10.4%; 48S, 61.2%). The percentage of complexes formed in the presence of cycloheximide were (C) CrPV IRES/cycloheximide (80S, <0.5%), CrPV IRES/cycloheximide + NSC119889 (80S, 8.0%) and (D) HCV IRES/cycloheximide (40S, 12.2%; 48S, 6.4%; and 80S, 69.2%), HCV IRES/cycloheximide + NSC119889 (40S, 12.5%; 48S, 10.5%; and 80S, 61.8%).