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

Most viral MTases perform cap-dependent N7 and/or 2'O methylations (Supplementary Figure S2A) (34, (48) (49) (50) (51) . However, it has recently been shown that flavivirus MTases (Dengue virus and Zika virus) additionally carry out internal A-2'O methylations (29, 34) . These activities might be related to the epitranscriptomic RNA modifications recently reported in the genome of flaviviruses (52) . Here, we report for the first time that internal A-2'O MTase activity also exists in viruses belonging to the Mononegavirales order (i.e. hMPV and SUDV) (Figure 2 and Supplementary Figure S3A ). The level of internal adenosine methylation obtained with the MTase+CTD of SUDV is clearly much higher than those obtained with hMPV, Zika and Dengue viruses, which predominantly target the cap structure. This higher level may be due to the fact that SUDV MTase is able to methylate A-2'O groups on both single-and double-stranded RNA structures, unlike ZIKV MTase, which carries out A-2'O methylations on singlestranded RNA only (Supplementary Figure S3B) . It is interesting to note that SUDV and hMPV MTase induces mainly internal A-2'O methylation as recently described for ZIKV and dengue virus MTase. This observation suggests that residues in the RNA binding site specifically recognize adenosines residues. In the case of dengue virus MTase, the molecular basis of the specific adenosine recognition at n1 position was elucidated. The crystal structure of the dengue virus MTase in presence of its RNA substrate indicate that the n1 adenosine base fits in a pocket shaped by residues I147-G148-E149-S150 downstream of the D146 catalytic residue (53) . The structure also reveals that G, in place of A residue would sterically clash with SAH and that pyrimidine (U or C) bases would decrease the van der Waals interactions with the protein explaining why the dengue virus MTase induces internal 2 -O-methylations on polyA but not on polyG, polyC, or polyU substrates (34) . Unfortunately, the residues downstream of the D catalytic residues are not conserved in SUDV MTase, and it is impossible to infer the residue involved in A1 recognition in the absence of structural data.