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

Based on bioinformatics analyses (Supplementary Figure S1 ), we designed several constructs encoding the C-terminal domains of SUDV L protein, and we tested their expression in bacteria. We selected the construct spanning amino-acids 1713-2211 ( Figure 1B) , which produced soluble MTase+CTD protein. After optimization of the solubilisation buffer, the SUDV MTase+CTD was purified to homogeneity as previously described for hMPV (26) (Figure 1C ). To characterize its MTase activity we first monitored the transfer by the SUDV MTase+CTD domain of radioactive methyl groups from the S-adenosylmethionine (SAM) methyldonor to a variety of capped RNAs mimicking the conserved 5 end of SUDV transcripts. Some of these RNAs contained non-radioactive methyl groups at key positions (i.e. the N7-position of the cap or the 2'Opositions of n1 or n2--see Supplementary Table S1 ), to help identify the positions to which the radiolabelled methyl groups were transferred ( Figure 2A) . Unexpectedly, we observed a strong MTase activity on cap-0 ( m GpppG), cap-1 ( m GpppG m ) and cap-2 ( m GpppG m A m ) RNAs. This activity was not observed with human RNA-N7 methyltransferase (RNMT) or the vaccinia virus 2'O MTase (VP39), whose activities result in m G and n1 m caps, respectively (Supplementary Figure S2A ). The methylation profile is also dif-ferent from that observed with the MTase+CTD domain of the closely related hMPV virus, which mainly targets the mRNA cap structure (Supplementary Figure S2B) and is weakly active on cap-1 RNA substrates. These observations thus suggest that the SUDV MTase+CTD methylates RNA at unconventional positions. To confirm the specificity of methylation observed, we mutated the conserved K-D-K-E catalytic residues typical for 2'O methyltransferases. Supplementary Figure S1 and Figure 2B demonstrated that MTase activity is strongly decreased by alanine mutation of each 2'O MTase catalytic residues. We then examined whether the presence of the cap was needed for this unconventional activity, and observed that both monophosphate and triphosphate RNAs could be methylated, although to a lower extent than the capped form, indicating cap-independent methylation in our experimental conditions ( Figure 2C ). This methylation does not seem to target the n1 as pppG m AU-SUDV 9 is also methylated. These observations are further supported by similar dissociation constants measured for binding to pppG-SUDV 12 and GpppG-SUDV 12 (641 and 682 nM, respectively), suggesting SUDV MTase+CTD targets sites outside the cap ( Figure 2D ). Since the MTase activity depends on the 2'O MTase catalytic tetrad ( Figure 2B ), we examined whether the methylation reaction targets 2'OH groups of nucleotides within the RNA.