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

To further characterize cap methylations, we used synthetic RNAs with internal 2'O-methylated adenosines and caps pre-methylated at different positions ( (m) GpppX (m) (A m )-SUDV 11 ). These RNAs mimicking the conserved start sequence of SUDV 5 transcripts and start with either an A or a G. Only the cap with a 2'O-methylated G (GpppG m ) at n1 was specifically methylated ( Figure 4A ), indicating that SUDV MTase+CTD has a cap N7 MTase activity, which is dependent on prior 2'O methylation of the n1 guanosine. This N7 MTase activity is pH-dependent with an optimal pH ranging between 7.0 and 7.5 whereas 2'O internal methylation peaks at a higher pH (Supplementary Figure S6) . We further characterized the nature of the methylation of the cap structure by TLC analysis ( Figure 4B ). In this time course experiment, 2'O-methylated RNAs with 32 Pradiolabeled caps were incubated with SUDV MTase+CTD before RNA digestion by P1 nuclease. The release of a cap-1 ( m GpppG m ) structure could be evidenced by TLC separation ( Figure 4B ). Altogether, these experiments demonstrate that the MTase+CTD domain of SUDV has cap N7-MTase activity on RNAs starting with a G. Interestingly, we did not detect N7 methylation using cap 2'O-methylated RNA beginning with an A (GpppA m ), suggesting substrate specificity based on the first nucleotide of the RNA. While this methylation requires a 2'O-methylated nucleotide at the first position of the RNA, cap 2'O methylation could not be detected in our enzymatic assays performed with short RNA substrate (VP40-13 mers). We thus evaluated capdependent 2'O MTase activity on synthetic RNA substrates of various lengths mimicking the 5 end of VP40 mRNA (Supplementary Tables S1 and S2). The short RNAs (VP40-13-mers) could not be methylated ( Figure 4C guanosine ( Figure 4C ), suggesting that RNA length or folding is a key factor for cap-dependent 2'O methylations.