Analysis of 3′SL RNA interaction with WT and mutant NS5 by coimmunoprecipitation and dynamic light scattering. (A) Western blot analysis showing pull-down of WT NS5, R888A mutant, and GAA mutant through 3′SL RNA immobilized on streptavidin Sepharose beads or the “no-RNA” mock coupled Sepharose beads as control. NS5 was detected by in-house 5M1 antibody (ref) which recognizes the MTase domain. (B) Quantification of pull-down WT, R888A, and GAA with the amount of WT normalized to 100%. The data were represented as average ± SD from two independent repeat experiments. The statistical significance of difference between the two groups was evaluated by Student's t-test and a P-value <0.05 was considered significant (≤0.0001, ≤0.001, ≤0.01, ≤0.05 were represented by [****], [***], [**], [*], respectively). (C,D) Intensity-based size distribution profiles from dynamic light scattering (DLS) experiments showing 3′SL RNA, NS5 WT, or molar ratio 1:1 mixture of NS5 and 3′SL complex (C) or R888A mutant (D) and the molar ratio 1:1 mixture of NS5 R888 and 3′SL. The DLS experiments were repeated twice with similar size distribution profiles for each sample.
