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

In agreement with previous studies on mononegavirus L proteins, the cap N7 methylation function was expected (22, 26, 35) . Our results indicate that this cap methylation occurs only if the first RNA nucleotide is a 2'O-methyl guanosine, like hMPV and VSV capping systems where the 2'O methylation of the first nucleotide precedes the N7 methylation of the cap guanosine (22, 26) . Nevertheless, we did not detect any N7 methylation of capped RNAs starting with a 2'O-methyl adenosine in our experimental system, suggesting substrate specificity based on the first nucleotide of the RNA. Since EBOV mRNAs start with an n1 guanosine (Dr Roland K. Hartmann, personal communication), this sequence specificity would ensure the specific methylation of SUDV mRNA. Contrary to what was observed with other viruses, such as coronaviruses (36, 37) or flaviviruses (38) (39) (40) , the 2'O methylation of the n1 residue in SUDV is barely detected on short RNA and requires RNAs longer than 30 nucleotides. Interestingly, the addition of the cap in VSV requires RNAs that are at least 31 nucleotides long (18) . These results prompt us to propose that transcription in the L protein starts off with the synthesis of a ∼30 nt-long uncapped RNA, which remains covalently bound to the PRN-Tase domain before the non-methylated cap guanosine is added. The RNA cap is then methylated at the 2'O position of n1 before N7 methyaltion of the cap guanosine by the MTase+CTD domain of L protein.