In vitro reconstitution of SARS-coronavirus mRNA cap methylation

PLoS Pathog. 2010 Apr 22;6(4):e1000863. doi: 10.1371/journal.ppat.1000863.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Exoribonucleases / chemistry
  • Exoribonucleases / genetics
  • Exoribonucleases / metabolism
  • Gene Expression Regulation, Viral
  • In Vitro Techniques
  • Methylation
  • RNA Caps / chemistry
  • RNA Caps / genetics*
  • RNA Caps / metabolism*
  • RNA, Messenger
  • Severe acute respiratory syndrome-related coronavirus / chemistry
  • Severe acute respiratory syndrome-related coronavirus / genetics*
  • Severe acute respiratory syndrome-related coronavirus / metabolism*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • tRNA Methyltransferases

Substances

  • RNA Caps
  • RNA, Messenger
  • Viral Nonstructural Proteins
  • tRNA Methyltransferases
  • nsp14 protein, SARS coronavirus
  • Exoribonucleases