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

Radioactive capped RNAs (G*pppRNA) were synthesized by incubating pppRNA (10 M) with vaccinia virus capping enzyme (New England Biolabs) in the presence of 1.65 mCi of [␣-32 P]-GTP (Perkin Elmer). Labeled RNAs were purified with StrataClean beads (Agilent) to remove proteins and G25 columns (GE Healthcare) to remove excess of radioactive GTP. RNA was then submitted to methylation by the SUDV MTase+CTD domain (as above), and precipitated as described for the in vitro transcription products. Finally, RNAs were digested with 1 U of nuclease P1 (Sigma) in 30 mM sodium acetate (pH 5.3), 5 mM ZnCl 2 and 50 mM NaCl (4 h, 37 • C). Products were spotted onto polyethylenimine cellulose thin-layer chromatography plates (Macherey-Nagel) and resolved using 0.65 M LiCl as mobile phase. The radiolabeled caps released by nuclease P1 were visualized using a Fluorescent Image Analyzer FLA3000 (Fuji) phosphor-imager.