The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
Identifieur interne : 001C41 ( Main/Exploration ); précédent : 001C40; suivant : 001C42The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
Auteurs : Aartjan J W. Te Velthuis [Pays-Bas] ; Sjoerd H E. Van Den Worm ; Eric J. SnijderSource :
- Nucleic acids research [ 1362-4962 ] ; 2012.
Descripteurs français
- KwdFr :
- ARN (métabolisme), ARN double brin (métabolisme), Cations divalents (), DNA-directed RNA polymerases (métabolisme), Données de séquences moléculaires, Motifs d'acides aminés, Multimérisation de protéines, Mutagenèse dirigée, Protons, Protéines virales (), Protéines virales (génétique), Protéines virales (métabolisme), RNA replicase (), RNA replicase (génétique), RNA replicase (métabolisme), Similitude de séquences d'acides aminés, Séquence d'acides aminés, Virus du SRAS (enzymologie).
- MESH :
- enzymologie : Virus du SRAS.
- génétique : Protéines virales, RNA replicase.
- métabolisme : ARN, ARN double brin, DNA-directed RNA polymerases, Protéines virales, RNA replicase.
- Cations divalents, Données de séquences moléculaires, Motifs d'acides aminés, Multimérisation de protéines, Mutagenèse dirigée, Protons, Protéines virales, RNA replicase, Similitude de séquences d'acides aminés, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Motifs, Amino Acid Sequence, Cations, Divalent (chemistry), DNA-Directed RNA Polymerases (metabolism), Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Multimerization, Protons, RNA (metabolism), RNA Replicase (chemistry), RNA Replicase (genetics), RNA Replicase (metabolism), RNA, Double-Stranded (metabolism), SARS Virus (enzymology), Sequence Homology, Amino Acid, Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Cations, Divalent, RNA Replicase, Viral Proteins.
- chemical , genetics : RNA Replicase, Viral Proteins.
- chemical , metabolism : DNA-Directed RNA Polymerases, RNA, RNA Replicase, RNA, Double-Stranded, Viral Proteins.
- enzymology : SARS Virus.
- Amino Acid Motifs, Amino Acid Sequence, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Multimerization, Protons, Sequence Homology, Amino Acid.
Abstract
Uniquely among RNA viruses, replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
DOI: 10.1093/nar/gkr893
PubMed: 22039154
Affiliations:
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Te Velthuis</name><affiliation wicri:level="1"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300RC Leiden, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300RC Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Van Den Worm, Sjoerd H E" sort="Van Den Worm, Sjoerd H E" uniqKey="Van Den Worm S" first="Sjoerd H E" last="Van Den Worm">Sjoerd H E. Van Den Worm</name></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. 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The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li></settlement></list><tree><noCountry><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name><name sortKey="Van Den Worm, Sjoerd H E" sort="Van Den Worm, Sjoerd H E" uniqKey="Van Den Worm S" first="Sjoerd H E" last="Van Den Worm">Sjoerd H E. Van Den Worm</name></noCountry><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="Te Velthuis, Aartjan J W" sort="Te Velthuis, Aartjan J W" uniqKey="Te Velthuis A" first="Aartjan J W" last="Te Velthuis">Aartjan J W. 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