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

By using various homopolymeric RNA substrates (27mers) , we demonstrated that the MTase+CTD preferentially methylates adenosine residues ( Figure 3A ) and is not active on poly G, C and U RNAs. Conversely, the MTase+CTD was unable to transfer methyl groups to the A 27 RNA (HO-(A m ) 27 ) substrate, of which all adenosines were pre-methylated at their 2'OH groups ( Figure 3B ). In addition, MTase activity is still detected using HO-(A m )A 26 and HO-(A m A m )A 25 RNAs as substrates. These results suggest that the SUDV MTase+CTD targets 2'O positions of adenosines within the RNA substrates downstream of the cap structure. This internal adenosine MTase activity appears to be the main activity of SUDV MTase in our experimental conditions. Conversely, the ratio of MTase activities on cap-1 RNAs (mGpppXm-RNA) over those on capped but unmethylated RNAs (GpppX-RNA) indicates that hMPV, ZIKV and DV MTases predominantly methylate the cap-structure ( Supplementary Figure S3A) . Additionally, SUDV MTase+CTD also methylates double-stranded RNAs, contrary to ZIKV MTase that only targets adenosines of single-stranded RNAs (Supplementary Figure S3B ) (29) . Further tests showed that heteropolymeric cap-1 RNAs mimicking the conserved start sequence of SUDV 5 transcripts are good MTase substrates even if their inner G or U residues were previously methylated ( Figure 3C ). Conversely, prior 2'O methylation of adenosine residues almost completely abolishes methyl transfer, confirming that there is no other unconventional activity on the cap structure. Methylation of internal residues does not decrease RNA/protein affinity as Kds for GpppG-SUDV 12 , m GpppG m (A m )-SUDV 11 , HO- Figure 3E ). After digestion, 98% of released residues correspond to native unmethylated adenosines and the remaining 2% were assigned to 2'O-methyl adenosines (Supplementary Figure S4) , which is consistent with the ratio of not transferred versus transferred tritiated methyl group during MTase assay (Supplementary Figure S5) . These data confirm the identification of an internal adenosine-specific 2'O methyltransferase activity (internal A-2'O MTase).