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

Pellets corresponding to 1 l of culture expressing SUDV MTase+CTD were thawed on ice, and bacteria lysed in a volume equal to 10 times final culture O.D. of optimized lysis buffer (1× BugBuster buffer (from a 10X solution, Merck) auditioned with 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 30 mM imidazole, 1 mM PMSF, 100 g/ml lysozyme, 1 g/ml DNase and 1% Triton X100). After clarification (45 000 × g, 30 min, 4 • C), lysates were incubated with CoNTA resin (Thermo; 0.5 ml/l culture) for 30 min at 4 • C, with gentle shaking. The beads were transferred to a 25 ml column and washed with 2 × 20 ml of buffer W1 (50 mM Tris pH 8, 1 M NaCl, 5% glycerol, 30 mM imidazole) and 10 ml of buffer W2 (50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 150 mM arginine). Proteins were eluted in buffer E (50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 1 M arginine). Finally, proteins were concentrated using Amicon Ultra (EMD Millipore) ultrafiltration units, and stored at -80 • C in 50% glycerol.