The methyltransferase domain of the Sudan ebolavirus L protein specifically targets internal adenosines of RNA substrates, in addition to the cap structure

Martin, Baptiste; Coutard, Bruno; Guez, Théo; Paesen, Guido C; Canard, Bruno; Debart, Françoise; Vasseur, Jean-Jacques; Grimes, Jonathan M; Decroly, Etienne

Using T4 RNA ligase 1 (20 units; New England Biolabs), cyanine 5-cytidine-5 -phosphate-3 -(6aminohexyl)phosphate (12.5 M, Jena Bioscience) was ligated to the 3 ends of the RNA substrates (10 M) in T4 RNA ligase 1 buffer (New England Biolabs), 1 mM ATP (16 • C, overnight). Ligase was removed by RNA precipitation in 3 M sodium acetate supplemented with glycogen (Thermo Scientific) (to 1 g/l). The fluorescent RNA was incubated (5 min at room temperature) with increasing concentrations of the SUDV MTase+CTD domain, in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol. Fluorescence polarization (FP) measurements were performed in a microplate reader (PHERAstar FS; BMG Labtech) with an optical module equipped with polarizers and using excitation and emission wavelengths of 590 and 675 nm, respectively. Dissociation constants (K d ) were determined using Hill slope curve fitting (Prism).