Figure 5.

Analysis of initiation complexes formed on the HCV IRES in the presence of NSC119889. (A) Agarose gel analysis of RNA isolated from 48S and 80S complexes purified by HCV IRES chromatography. 48S and 80S complexes were formed on the HCV IRES as described in Materials and Methods in the presence of GMP-PNP or cycloheximide, respectively. RNA was isolated from these complexes, fractionated on a 1% agarose/formaldehyde, and visualized by staining with ethidium bromide. (B) Western blot detection of eIF2α and the p116 subunit of eIF3 in initiation complexes formed on the HCV IRES. Similar cell equivalents from the 48S and 80S complexes were TCA precipitated, fractionated by SDS-PAGE, and transferred to Immobilon P (Millipore) membrane. Immunoblots were performed with the indicated antibodies. The origin of each sample is indicated above the panel. (C) Detection of Met-tRNA_i in the 48S and 80S initiation complexes. RNA was isolated from fractions containing 48S and 80S complexes using TRIzol, following the manufacturer's recommendation (Invitrogen). Five micrograms, representing 53 and 33% of the RNA recovered from 48S and 80S fractions, respectively, was analyzed on a 8 M urea/10% polyacrylamide gel and transferred onto a Hybond-N⁺ membrane (GE Healthcare) using a Transblot SD semidry apparatus (Bio-Rad). The RNA was cross-linked using a UV-Stratalinker 2400 (Stratagene) and tRNA_i^Met detected by Northern blotting using a DNA oligonucleotide targeting mammalian tRNAi^Met. (D) The HCV IRES uses Met-tRNA_i recruited in the presence of NSC119889 for translation. Translations were performed in Krebs-2 extracts in the presence of unlabeled methionine and supplemented with in vitro charged [³⁵S]Met-tRNA_i^Met at the indicated concentrations of NSC119889. Translations were programmed with 10 μg/ml FF/HCV/Ren mRNA. Products were electrophoresed on a 10% SDS-polyacrylamide gel, treated with EN³Hance, dried, and exposed to X-OMAT film (Eastman Kodak).