Identification of a Nidovirales Orf1a N7-guanine cap Methyltransferase signature-sequence as a genetic marker of large genome Tobaniviridae

François Ferron; Humberto Julio Debat; Etienne Decroly; Bruno Canard

Unlike 2'-O MTases and their well-defined K-D-K-E tetrad, and apart from NRF nsp14-like MTases, signature sequences of RNA cap N7-guanine cap N7-guanosine MTases are much less obvious. The wealth of structural data and mechanistic insight on the N7-guanine MTases enzymes has been acquired from the microsporidian parasite Encephalitozoon cuniculi [36] , the poxvirus MTase D1:D12 heterodimer [37] and Reoviridae [38, 39] in the case of RNA viruses. There are only three crystal structure of RF-MTase known to methylate N7-guanine RNA caps of RNA viruses : those of Reoviridae (Rotavirus VP4 and Reovirus Lambda2), and Flavivirus, whose NS5 N-terminus domain is a bi-functional N7-guanine and 2'O-MTase [40] . Taking together these data, and combining structural information with the human RNA N7-guanine methyltransferase crystal structure (PDB ID: 3BGV), one cannot define a specific N7-guanine MTase signature (Fig. S1 ). From this analysis and others [35] it is clear that the structure conservation prevailed over sequence conservation. No amino acid motifs typical of an RNA N7-guanine MTase can be clearly identified (Fig. S1) . A narrower structural comparison including only Encephalitozoon cuniculi, poxvirus and the human RNA N7-guanine MTase allows detection of 6 amino acids or motifs K / G/D/ HY / E / Y (Fig. S2 ). Typical SAM binding motifs are defined by a combination of 3 motifs placed at specific structural positions: K within an α helix, followed by a GxGxG motif within a loop and conserved D. The 3 latter motifs are located in the MTase structural pocket where the N7 methylation supposedly occurs. In order to use an independent method, we performed a structural search using HHpred on PDB and SCOPe databases [41] , using each sequence as a query. This allowed to refine the boundaries of the MTase domain. The structure with the closest hit is a SAM-dependent MTase from Pectobacterium atrosepticum and then Encephalitozoon cuniculi. A quick superimposition of the retrieved structures with N7-guanine MTase shows that these structures can be structurally aligned but with very limited sequence conservation. Surprisingly, apart from a conserved leucine residue 2 amino acid upstream the poorly conserved SAM binding motif (only the third G is strictly conserved) and a glycine-rich motifs in the fifth beta sheet, nothing else is conserved nor defines an active site (Fig. S3 ). Taking these criteria into account, there was no obvious indication of RF N7-guanine MTase features in Nidovirales other than the two mentioned above in Abyssoviridae and Mononiviridae. Currently, based on the detection of RF-MTase fold, one can infer a 2' O-MTase sequence signature when the K-D-K-E tetrad is present, but not a RNA-cap N7guanine MTase.