Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis.
Identifieur interne : 002219 ( PubMed/Checkpoint ); précédent : 002218; suivant : 002220Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis.
Auteurs : Ekaterina Minskaia [Allemagne] ; Tobias Hertzig ; Alexander E. Gorbalenya ; Valérie Campanacci ; Christian Cambillau ; Bruno Canard ; John ZiebuhrSource :
- Proceedings of the National Academy of Sciences of the United States of America [ 0027-8424 ] ; 2006.
Descripteurs français
- KwdFr :
- ARN double brin (métabolisme), ARN viral (biosynthèse), ARN viral (génétique), Alignement de séquences, Cations divalents (), Coronavirus (enzymologie), Coronavirus (génétique), Coronavirus (physiologie), Données de séquences moléculaires, Exoribonucleases (), Exoribonucleases (génétique), Exoribonucleases (métabolisme), Métaux (), Métaux (pharmacologie), Noyau de la cellule (enzymologie), Noyau de la cellule (génétique), Réplication virale, Spécificité du substrat, Séquence conservée, Séquence d'acides aminés, Transcription génétique (génétique).
- MESH :
- biosynthèse : ARN viral.
- enzymologie : Coronavirus, Noyau de la cellule.
- génétique : ARN viral, Coronavirus, Exoribonucleases, Noyau de la cellule, Transcription génétique.
- métabolisme : ARN double brin, Exoribonucleases.
- pharmacologie : Métaux.
- physiologie : Coronavirus.
- Alignement de séquences, Cations divalents, Données de séquences moléculaires, Exoribonucleases, Métaux, Réplication virale, Spécificité du substrat, Séquence conservée, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Cations, Divalent (chemistry), Cell Nucleus (enzymology), Cell Nucleus (genetics), Conserved Sequence, Coronavirus (enzymology), Coronavirus (genetics), Coronavirus (physiology), Exoribonucleases (chemistry), Exoribonucleases (genetics), Exoribonucleases (metabolism), Metals (chemistry), Metals (pharmacology), Molecular Sequence Data, RNA, Double-Stranded (metabolism), RNA, Viral (biosynthesis), RNA, Viral (genetics), Sequence Alignment, Substrate Specificity, Transcription, Genetic (genetics), Virus Replication.
- MESH :
- chemical , biosynthesis : RNA, Viral.
- chemical , chemistry : Cations, Divalent, Exoribonucleases, Metals.
- enzymology : Cell Nucleus, Coronavirus.
- genetics : Cell Nucleus, Coronavirus, Exoribonucleases, RNA, Viral, Transcription, Genetic.
- chemical , metabolism : Exoribonucleases, RNA, Double-Stranded.
- chemical , pharmacology : Metals.
- physiology : Coronavirus.
- Amino Acid Sequence, Conserved Sequence, Molecular Sequence Data, Sequence Alignment, Substrate Specificity, Virus Replication.
Abstract
Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.
DOI: 10.1073/pnas.0508200103
PubMed: 16549795
Affiliations:
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pubmed:16549795Le document en format XML
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<front><div type="abstract" xml:lang="en">Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.</div>
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