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 replicas...

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Autores principales:	Mickaël Bouvet, Claire Debarnot, Isabelle Imbert, Barbara Selisko, Eric J Snijder, Bruno Canard, Etienne Decroly
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/29ecdc4d55f1477cb1101458d14f403f
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spelling	oai:doaj.org-article:29ecdc4d55f1477cb1101458d14f403f2021-11-25T05:48:07ZIn vitro reconstitution of SARS-coronavirus mRNA cap methylation.1553-73661553-737410.1371/journal.ppat.1000863https://doaj.org/article/29ecdc4d55f1477cb1101458d14f403f2010-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20421945/pdf/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374SARS-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.Mickaël BouvetClaire DebarnotIsabelle ImbertBarbara SeliskoEric J SnijderBruno CanardEtienne DecrolyPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 6, Iss 4, p e1000863 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Mickaël Bouvet Claire Debarnot Isabelle Imbert Barbara Selisko Eric J Snijder Bruno Canard Etienne Decroly In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
description	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.
format	article
author	Mickaël Bouvet Claire Debarnot Isabelle Imbert Barbara Selisko Eric J Snijder Bruno Canard Etienne Decroly
author_facet	Mickaël Bouvet Claire Debarnot Isabelle Imbert Barbara Selisko Eric J Snijder Bruno Canard Etienne Decroly
author_sort	Mickaël Bouvet
title	In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
title_short	In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
title_full	In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
title_fullStr	In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
title_full_unstemmed	In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
title_sort	in vitro reconstitution of sars-coronavirus mrna cap methylation.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/29ecdc4d55f1477cb1101458d14f403f
work_keys_str_mv	AT mickaelbouvet invitroreconstitutionofsarscoronavirusmrnacapmethylation AT clairedebarnot invitroreconstitutionofsarscoronavirusmrnacapmethylation AT isabelleimbert invitroreconstitutionofsarscoronavirusmrnacapmethylation AT barbaraselisko invitroreconstitutionofsarscoronavirusmrnacapmethylation AT ericjsnijder invitroreconstitutionofsarscoronavirusmrnacapmethylation AT brunocanard invitroreconstitutionofsarscoronavirusmrnacapmethylation AT etiennedecroly invitroreconstitutionofsarscoronavirusmrnacapmethylation
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In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

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