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

Each HO-(A) 27 , HO-(A m ) 27 and HO-(A) 27 RNA which was preliminarily treated with the SUDV Mtase+CTD domain Nucleic Acids Research, 2018, Vol. 46, No. 15 7905 (2.5 g/l) in buffer (50 mM Tris-HCl pH 7.5 containing 50 mM NaCl, 10 mM MgCl 2 ; final concentrations) was incubated with snake venom phosphodiesterase (0.2 unit/mol RNA) at 37 • C for 15 h. Then alkaline phosphatase (150 units/mol RNA) was added and the mixture was incubated at 37 • C for a further 30 min. The resulting digestion mixture was then analyzed by reverse-phase HPLC (column Macherey-Nagel Nucleodur C 18 , 7.5 × 4.6 mm, 100-3 EC, flow rate: 1 ml/min). Elution was performed with a 20 min linear gradient of 8% of acetonitrile in 50 mM triethylammonium acetate, pH 7. UV detection was carried out at 260 nm.