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 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.
Bouvet
Mickaël
M
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, Marseille, France.
Debarnot
Claire
C
Imbert
Isabelle
I
Selisko
Barbara
B
Snijder
Eric J
EJ
Canard
Bruno
B
Decroly
Etienne
E
D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D008745:Methylation; D012315:RNA Caps / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012359:tRNA Methyltransferases
D012315:RNA Caps; D012333:RNA, Messenger; D017361:Viral Nonstructural Proteins; D012359:tRNA Methyltransferases; C525596:nsp14 protein, SARS coronavirus; D005095:Exoribonucleases