Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity.
Identifieur interne : 001C92 ( Main/Exploration ); précédent : 001C91; suivant : 001C93Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity.
Auteurs : Yibei Xiao [Allemagne] ; Qingjun Ma ; Tobias Restle ; Weifeng Shang ; Dmitri I. Svergun ; Rajesh Ponnusamy ; Georg Sczakiel ; Rolf HilgenfeldSource :
- Journal of virology [ 1098-5514 ] ; 2012.
Descripteurs français
- KwdFr :
- Alignement de séquences, Animaux, Conformation des protéines, Coronavirus félin (), Coronavirus félin (enzymologie), DNA-directed RNA polymerases (), DNA-directed RNA polymerases (métabolisme), Données de séquences moléculaires, Liaison aux protéines, Modèles moléculaires, Multimérisation de protéines, Protéines virales non structurales (), Protéines virales non structurales (métabolisme), Séquence d'acides aminés.
- MESH :
- enzymologie : Coronavirus félin.
- métabolisme : DNA-directed RNA polymerases, Protéines virales non structurales.
- Alignement de séquences, Animaux, Conformation des protéines, Coronavirus félin, DNA-directed RNA polymerases, Données de séquences moléculaires, Liaison aux protéines, Modèles moléculaires, Multimérisation de protéines, Protéines virales non structurales, Séquence d'acides aminés.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Coronavirus, Feline (chemistry), Coronavirus, Feline (enzymology), DNA-Directed RNA Polymerases (chemistry), DNA-Directed RNA Polymerases (metabolism), Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Multimerization, Sequence Alignment, Viral Nonstructural Proteins (chemistry), Viral Nonstructural Proteins (metabolism).
- MESH :
- chemical , chemistry : DNA-Directed RNA Polymerases, Viral Nonstructural Proteins.
- chemistry : Coronavirus, Feline.
- enzymology : Coronavirus, Feline.
- chemical , metabolism : DNA-Directed RNA Polymerases, Viral Nonstructural Proteins.
- Amino Acid Sequence, Animals, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Multimerization, Sequence Alignment.
Abstract
Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/β domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring the sequence GPLG instead of the His(6) tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.
DOI: 10.1128/JVI.06635-11
PubMed: 22318142
Affiliations:
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Le document en format XML
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Svergun</name></author><author><name sortKey="Ponnusamy, Rajesh" sort="Ponnusamy, Rajesh" uniqKey="Ponnusamy R" first="Rajesh" last="Ponnusamy">Rajesh Ponnusamy</name></author><author><name sortKey="Sczakiel, Georg" sort="Sczakiel, Georg" uniqKey="Sczakiel G" first="Georg" last="Sczakiel">Georg Sczakiel</name></author><author><name sortKey="Hilgenfeld, Rolf" sort="Hilgenfeld, Rolf" uniqKey="Hilgenfeld R" first="Rolf" last="Hilgenfeld">Rolf Hilgenfeld</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2012">2012</date><idno type="RBID">pubmed:22318142</idno><idno type="pmid">22318142</idno><idno type="doi">10.1128/JVI.06635-11</idno><idno type="wicri:Area/PubMed/Corpus">001404</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001404</idno><idno type="wicri:Area/PubMed/Curation">001404</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001404</idno><idno type="wicri:Area/PubMed/Checkpoint">001320</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">001320</idno><idno type="wicri:Area/Ncbi/Merge">002457</idno><idno type="wicri:Area/Ncbi/Curation">002457</idno><idno type="wicri:Area/Ncbi/Checkpoint">002457</idno><idno type="wicri:Area/Main/Merge">001D08</idno><idno type="wicri:Area/Main/Curation">001C92</idno><idno type="wicri:Area/Main/Exploration">001C92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity.</title><author><name sortKey="Xiao, Yibei" sort="Xiao, Yibei" uniqKey="Xiao Y" first="Yibei" last="Xiao">Yibei Xiao</name><affiliation wicri:level="1"><nlm:affiliation>Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck</wicri:regionArea><wicri:noRegion>Lübeck</wicri:noRegion><wicri:noRegion>Lübeck</wicri:noRegion><wicri:noRegion>Lübeck</wicri:noRegion></affiliation></author><author><name sortKey="Ma, Qingjun" sort="Ma, Qingjun" uniqKey="Ma Q" first="Qingjun" last="Ma">Qingjun Ma</name></author><author><name sortKey="Restle, Tobias" sort="Restle, Tobias" uniqKey="Restle T" first="Tobias" last="Restle">Tobias Restle</name></author><author><name sortKey="Shang, Weifeng" sort="Shang, Weifeng" uniqKey="Shang W" first="Weifeng" last="Shang">Weifeng Shang</name></author><author><name sortKey="Svergun, Dmitri I" sort="Svergun, Dmitri I" uniqKey="Svergun D" first="Dmitri I" last="Svergun">Dmitri I. Svergun</name></author><author><name sortKey="Ponnusamy, Rajesh" sort="Ponnusamy, Rajesh" uniqKey="Ponnusamy R" first="Rajesh" last="Ponnusamy">Rajesh Ponnusamy</name></author><author><name sortKey="Sczakiel, Georg" sort="Sczakiel, Georg" uniqKey="Sczakiel G" first="Georg" last="Sczakiel">Georg Sczakiel</name></author><author><name sortKey="Hilgenfeld, Rolf" sort="Hilgenfeld, Rolf" uniqKey="Hilgenfeld R" first="Rolf" last="Hilgenfeld">Rolf Hilgenfeld</name></author></analytic><series><title level="j">Journal of virology</title><idno type="eISSN">1098-5514</idno><imprint><date when="2012" type="published">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>Coronavirus, Feline (chemistry)</term><term>Coronavirus, Feline (enzymology)</term><term>DNA-Directed RNA Polymerases (chemistry)</term><term>DNA-Directed RNA Polymerases (metabolism)</term><term>Models, Molecular</term><term>Molecular Sequence Data</term><term>Protein Binding</term><term>Protein Conformation</term><term>Protein Multimerization</term><term>Sequence Alignment</term><term>Viral Nonstructural Proteins (chemistry)</term><term>Viral Nonstructural Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Alignement de séquences</term><term>Animaux</term><term>Conformation des protéines</term><term>Coronavirus félin ()</term><term>Coronavirus félin (enzymologie)</term><term>DNA-directed RNA polymerases ()</term><term>DNA-directed RNA polymerases (métabolisme)</term><term>Données de séquences moléculaires</term><term>Liaison aux protéines</term><term>Modèles moléculaires</term><term>Multimérisation de protéines</term><term>Protéines virales non structurales ()</term><term>Protéines virales non structurales (métabolisme)</term><term>Séquence d'acides aminés</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA-Directed RNA Polymerases</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Coronavirus, Feline</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Coronavirus félin</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Coronavirus, Feline</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA-Directed RNA Polymerases</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>DNA-directed RNA polymerases</term><term>Protéines virales non structurales</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>Models, Molecular</term><term>Molecular Sequence Data</term><term>Protein Binding</term><term>Protein Conformation</term><term>Protein Multimerization</term><term>Sequence Alignment</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Alignement de séquences</term><term>Animaux</term><term>Conformation des protéines</term><term>Coronavirus félin</term><term>DNA-directed RNA polymerases</term><term>Données de séquences moléculaires</term><term>Liaison aux protéines</term><term>Modèles moléculaires</term><term>Multimérisation de protéines</term><term>Protéines virales non structurales</term><term>Séquence d'acides aminés</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His(6)-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/β domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His(6) tag. Remarkably, the same protein harboring the sequence GPLG instead of the His(6) tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.</div></front></TEI><affiliations><list><country><li>Allemagne</li></country></list><tree><noCountry><name sortKey="Hilgenfeld, Rolf" sort="Hilgenfeld, Rolf" uniqKey="Hilgenfeld R" first="Rolf" last="Hilgenfeld">Rolf Hilgenfeld</name><name sortKey="Ma, Qingjun" sort="Ma, Qingjun" uniqKey="Ma Q" first="Qingjun" last="Ma">Qingjun Ma</name><name sortKey="Ponnusamy, Rajesh" sort="Ponnusamy, Rajesh" uniqKey="Ponnusamy R" first="Rajesh" last="Ponnusamy">Rajesh Ponnusamy</name><name sortKey="Restle, Tobias" sort="Restle, Tobias" uniqKey="Restle T" first="Tobias" last="Restle">Tobias Restle</name><name sortKey="Sczakiel, Georg" sort="Sczakiel, Georg" uniqKey="Sczakiel G" first="Georg" last="Sczakiel">Georg Sczakiel</name><name sortKey="Shang, Weifeng" sort="Shang, Weifeng" uniqKey="Shang W" first="Weifeng" last="Shang">Weifeng Shang</name><name sortKey="Svergun, Dmitri I" sort="Svergun, Dmitri I" uniqKey="Svergun D" first="Dmitri I" last="Svergun">Dmitri I. Svergun</name></noCountry><country name="Allemagne"><noRegion><name sortKey="Xiao, Yibei" sort="Xiao, Yibei" uniqKey="Xiao Y" first="Yibei" last="Xiao">Yibei Xiao</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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