FIG. 6.

G13 and U14 in the NS1 5′ UTR are required for GRSF-1 binding. Wild-type (Wt) GST-GRSF-1 (1.25 × 10⁻¹¹ mol) was incubated with 10⁶ dpm of radiolabeled wild-type NS1 5′ UTR RNA in the absence or presence of increasing concentrations (10- or 100-fold molar excess) of unlabeled wild-type (lanes 2 to 4), U14C (lanes 5 to 7), or G13C/U14C (lanes 8 to 10) competitor RNAs in 5 mM HEPES (pH 7.6)-25 mM KCl-2 mM MgCl₂-5% glycerol-100 mM NaCl-2 mM dithiothreitol-20 U of RNasin (Promega) at 30°C for 15 min. Samples were then exposed to UV light (1.0 J) in a Stratalinker (Stratagene), followed by incubation with 10 mg of RNase A per ml and 2 U of RNase T₁ at 37°C for 30 min. Samples were suspended in 1× Laemmli buffer and separated by SDS-10% PAGE, and the gel was dried and visualized with a Storm 850 phosphorimager (Molecular Dynamics). These data are representative of three independent experiments.