In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation

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 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.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Coronaviruses follow the canonical capping pathway, which consists of four sequential enzymatic reactions: ( a ) RTPase, encoded by the nsp13 helicase, hydrolyzing the γ-phosphate of the mRNA; ( b ) an as-yet-unidentified guanylyltransferase (GTase) adding GMP to the 5′-diphosphate RNA; ( c ) nsp14 N7-MTase methylating the guanosine, leading to a cap-0 structure that is essential for efficient translation initiation; and ( d ) nsp16 2′- O -methyltransferase (2′- O -MTase) carrying out further methylations, leading to cap-1 and cap-2 structures, which are required to efficiently escape the nonself RNA recognition system of the host cell (156). Interestingly, whereas nsp10 binding has no effect on nsp14 N7-MTase activity, nsp10 is required for nsp16 2- O -MTase activity.…”

Nidovirus ribonucleases: Structures and functions in viral replication

“…71,72 Though sequence similarity with other N7-MTs is low, a conserved motif with similarities to motif I of S-adenosyl methionine-dependent methyltransferases, the motif involved in S-adenosyl methionine binding, was identified C-proximal to the conserved ExoN domain. This C-terminal part of nsp14 was predicted to possess a canonical Rossmann fold.…”

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

“…The 2ЈO-MTase Activity of nsp16 Correlates with Its Interaction with nsp10 Mutants-SARS-CoV nsp10 was recently found to be a nsp16 helper protein; nsp10 turns on the otherwise inactive 2ЈO-MTase activity of nsp16 (1). We therefore analyzed the functional consequences of nsp10 mutations on nsp16 2ЈO-MTase activity.…”

“…Cap 1 structure formation requires an additional 2ЈO-MTase, that methylates the ribose 2ЈO-position of the first nucleotide of the mRNA. The involvement of nsp14 N7-MTase and of nsp16 2ЈO-MTase in the capping pathway was recently demonstrated biochemically (1,18,20). Moreover, both nsp14 and -16 play crucial roles for efficient RNA synthesis within the SARS-CoV replicon and for transcription/replication of MHV-CoV (13,24).…”