In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation: e1000863

• Published in: PLoS pathogens 2010-04, Vol.6 (4)
• Main Authors: Bouvet, Mickaël, Debarnot, Claire, Imbert, Isabelle, Selisko, Barbara, Snijder, Eric J, Canard, Bruno, Decroly, Etienne
• Format: Article
• Language: English
• Subjects: Enzymes; Experiments; Genomes; Proteins; RNA polymerase
• ISSN: 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1000863

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2'OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design.