Fig. 1.

ATP hydrolysis increases the binding preference of MDA5 for longer dsRNA. (A) EMSA of MDA5 and RIG-I with increasing amounts of 112-bp dsRNA. Protein (0.3 μM) was incubated with 112-bp dsRNA [30 nM (lane 1)–150 nM (lane 5)]. 3′-Fluorescein-labeled dsRNA was maintained at 10 nM, and the ratio of protein to binding sites (BS) was calculated assuming that each monomer occupied 14 bp. (B) Representative electron micrographs of MDA5 in complex with 2,012-bp dsRNA at a protein-to-binding site ratio of 1.8 (Left) or 0.31 (Right) in the presence of 0.5 mM ADP⋅AlF₄. (C) EMSA and representative class averages of filaments formed by the CARD deletion mutant MDA5h on 112-bp dsRNA in the presence of 0.5 mM ADP⋅AlF₄. EMSA was performed as in A. (D) Competition binding assay (mean ± SD, n = 2–3). Fluorescein-labeled reporter dsRNA (112 bp, 0.18 μg/mL) and unlabeled competitor dsRNA (21–2,012 bp, 0.06–43.74 μg/mL) were premixed and incubated with MDA5 (80 nM). The level of the reporter complex was monitored by EMSA in the presence of increasing amounts of competitor dsRNA (Fig. S2) and was plotted with fitted competition binding curves, which yielded the IC₅₀. (E) Competition EMSA of MDA5 with dsRNAs of various lengths with ADPCP or ATP (2 mM). Experiments were performed as in A with a fixed concentration of competitor dsRNAs (0.54 μg/mL). The relative level of labeled complex with respect to 62-bp competitor dsRNA was plotted (mean ± SD, n = 3). (F) Competition EMSA of RIG-I as in B (mean ± SD, n = 3). The two bands corresponding to RIG-I:RNA complexes with ADPCP suggest binding to each end of dsRNA. The presence of multiple ill-defined bands with ATP is consistent with the translocation of RIG-I on dsRNA (37).