In vitro reconstitution of sars-coronavirus mRNA cap methylation

Sars-coronavirus (sars-cov) genome expression depends on the synthesis of a set of mrnas,which presumably are capped at their 59 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. © 2010 bouvet et al.