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 7Me GpppA-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 7Me GpppA 2′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 IC 50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design. Author Summary In 2003, an emerging coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS). SARS-CoV replicates and transcribes its large RNA genome using a membrane-bound enzyme complex containing a variety of viral nonstructural proteins. A critical step during RNA synthesis is the addition of a cap structure to the newly produced viral mRNAs, ensuring their efficient translation by host cell ribosomes. Viruses generally acquire their cap structure either from cellular mRNAs (e.g., “cap snatching” of influenza virus) or employ their own capping machinery, as is supposed to be the case for coronaviruses. mRNA caps synthesized by viruses are structurally and functionally undistinguishable from cellular mRNAs caps. In coronaviruses, methylation of mRNA caps seems to be essential, since mutations in viral methyltransferases nsp14 or nsp16 render non-viable virus. We have discovered an unexpected key role for SARS-CoV nsp10, a protein of previously unknown function, within mRNA cap methylation. Nsp10 induces selective 2′O-methylation of guanine-N7 methylated capped RNAs through direct activation of the otherwise inactive nsp16. This finding allows the full reconstitution of the SARS-CoV mRNA cap methylation sequence in vitro and opens the way to exploit the mRNA cap methyltransferases as targets for anti-coronavirus drug design.
【초록키워드】 Coronaviruses, coronavirus, Mutation, drug design, translation, SARS-CoV, Genome, Influenza virus, in vitro, virus, RNA, activity, Protein, mRNA, methyltransferase, nsp14, non-structural protein, CoV, target, expression, Critical, Methylation, mRNAs, host cell, SARS-coronavirus, acute respiratory syndrome, Activation, Viral protein, enzyme, Author, RNA synthesis, complex, sequence, etiological agent, RNA genome, ribosomes, selective, infected cell, viral nonstructural proteins, membrane-bound, viral mRNAs, CAP, Complete, inhibition assay, event, produced, predicted, 2003, include, involved, addition, generate, replicate, in viral, variety, induce, initiated, inactive, RNA transcript, SARS-CoV infected cells, cellular mRNA, methylated, S-adenosyl-L-methionine,