The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
Identifieur interne : 005D38 ( Main/Exploration ); précédent : 005D37; suivant : 005D39The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
Auteurs : Julian A. Tanner [République populaire de Chine] ; Rory M. Watt ; Yu-Bo Chai ; Lin-Yu Lu ; Marie C. Lin ; J S Malik Peiris ; Leo L M. Poon ; Hsiang-Fu Kung ; Jian-Dong HuangSource :
- The Journal of biological chemistry [ 0021-9258 ] ; 2003.
Descripteurs français
- KwdFr :
- ADN viral (génétique), ADN viral (métabolisme), Animaux, Cellules Vero, Cinétique, Données de séquences moléculaires, Helicase (), Helicase (génétique), Helicase (métabolisme), Nucleoside-triphosphatase (), Nucleoside-triphosphatase (génétique), Nucleoside-triphosphatase (métabolisme), RNA helicases (), RNA helicases (génétique), RNA helicases (métabolisme), Spécificité du substrat, Séquence nucléotidique, Virus du SRAS (enzymologie), Virus du SRAS (génétique).
- MESH :
- enzymologie : Virus du SRAS.
- génétique : ADN viral, Helicase, Nucleoside-triphosphatase, RNA helicases, Virus du SRAS.
- métabolisme : ADN viral, Helicase, Nucleoside-triphosphatase, RNA helicases.
- Animaux, Cellules Vero, Cinétique, Données de séquences moléculaires, Helicase, Nucleoside-triphosphatase, RNA helicases, Spécificité du substrat, Séquence nucléotidique.
English descriptors
- KwdEn :
- Animals, Base Sequence, Chlorocebus aethiops, DNA Helicases (classification), DNA Helicases (genetics), DNA Helicases (metabolism), DNA, Viral (genetics), DNA, Viral (metabolism), Kinetics, Molecular Sequence Data, Nucleoside-Triphosphatase (classification), Nucleoside-Triphosphatase (genetics), Nucleoside-Triphosphatase (metabolism), RNA Helicases (classification), RNA Helicases (genetics), RNA Helicases (metabolism), SARS Virus (enzymology), SARS Virus (genetics), Substrate Specificity, Vero Cells.
- MESH :
- chemical , classification : DNA Helicases, Nucleoside-Triphosphatase, RNA Helicases.
- chemical , genetics : DNA Helicases, DNA, Viral, Nucleoside-Triphosphatase, RNA Helicases.
- chemical , metabolism : DNA Helicases, DNA, Viral, Nucleoside-Triphosphatase, RNA Helicases.
- enzymology : SARS Virus.
- genetics : SARS Virus.
- Animals, Base Sequence, Chlorocebus aethiops, Kinetics, Molecular Sequence Data, Substrate Specificity, Vero Cells.
Abstract
The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.
DOI: 10.1074/jbc.C300328200
PubMed: 12917423
Affiliations:
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Watt</name></author><author><name sortKey="Chai, Yu Bo" sort="Chai, Yu Bo" uniqKey="Chai Y" first="Yu-Bo" last="Chai">Yu-Bo Chai</name></author><author><name sortKey="Lu, Lin Yu" sort="Lu, Lin Yu" uniqKey="Lu L" first="Lin-Yu" last="Lu">Lin-Yu Lu</name></author><author><name sortKey="Lin, Marie C" sort="Lin, Marie C" uniqKey="Lin M" first="Marie C" last="Lin">Marie C. Lin</name></author><author><name sortKey="Peiris, J S Malik" sort="Peiris, J S Malik" uniqKey="Peiris J" first="J S Malik" last="Peiris">J S Malik Peiris</name></author><author><name sortKey="Poon, Leo L M" sort="Poon, Leo L M" uniqKey="Poon L" first="Leo L M" last="Poon">Leo L M. Poon</name></author><author><name sortKey="Kung, Hsiang Fu" sort="Kung, Hsiang Fu" uniqKey="Kung H" first="Hsiang-Fu" last="Kung">Hsiang-Fu Kung</name></author><author><name sortKey="Huang, Jian Dong" sort="Huang, Jian Dong" uniqKey="Huang J" first="Jian-Dong" last="Huang">Jian-Dong Huang</name></author></analytic><series><title level="j">The Journal of biological chemistry</title><idno type="ISSN">0021-9258</idno><imprint><date when="2003" type="published">2003</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Chlorocebus aethiops</term><term>DNA Helicases (classification)</term><term>DNA Helicases (genetics)</term><term>DNA Helicases (metabolism)</term><term>DNA, Viral (genetics)</term><term>DNA, Viral (metabolism)</term><term>Kinetics</term><term>Molecular Sequence Data</term><term>Nucleoside-Triphosphatase (classification)</term><term>Nucleoside-Triphosphatase (genetics)</term><term>Nucleoside-Triphosphatase (metabolism)</term><term>RNA Helicases (classification)</term><term>RNA Helicases (genetics)</term><term>RNA Helicases (metabolism)</term><term>SARS Virus (enzymology)</term><term>SARS Virus (genetics)</term><term>Substrate Specificity</term><term>Vero Cells</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN viral (génétique)</term><term>ADN viral (métabolisme)</term><term>Animaux</term><term>Cellules Vero</term><term>Cinétique</term><term>Données de séquences moléculaires</term><term>Helicase ()</term><term>Helicase (génétique)</term><term>Helicase (métabolisme)</term><term>Nucleoside-triphosphatase ()</term><term>Nucleoside-triphosphatase (génétique)</term><term>Nucleoside-triphosphatase (métabolisme)</term><term>RNA helicases ()</term><term>RNA helicases (génétique)</term><term>RNA helicases (métabolisme)</term><term>Spécificité du substrat</term><term>Séquence nucléotidique</term><term>Virus du SRAS (enzymologie)</term><term>Virus du SRAS (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="classification" xml:lang="en"><term>DNA Helicases</term><term>Nucleoside-Triphosphatase</term><term>RNA Helicases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Helicases</term><term>DNA, Viral</term><term>Nucleoside-Triphosphatase</term><term>RNA Helicases</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA Helicases</term><term>DNA, Viral</term><term>Nucleoside-Triphosphatase</term><term>RNA Helicases</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN viral</term><term>Helicase</term><term>Nucleoside-triphosphatase</term><term>RNA helicases</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ADN viral</term><term>Helicase</term><term>Nucleoside-triphosphatase</term><term>RNA helicases</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Chlorocebus aethiops</term><term>Kinetics</term><term>Molecular Sequence Data</term><term>Substrate Specificity</term><term>Vero Cells</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules Vero</term><term>Cinétique</term><term>Données de séquences moléculaires</term><term>Helicase</term><term>Nucleoside-triphosphatase</term><term>RNA helicases</term><term>Spécificité du substrat</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.</div></front></TEI><affiliations><list><country><li>République populaire de Chine</li></country></list><tree><noCountry><name sortKey="Chai, Yu Bo" sort="Chai, Yu Bo" uniqKey="Chai Y" first="Yu-Bo" last="Chai">Yu-Bo Chai</name><name sortKey="Huang, Jian Dong" sort="Huang, Jian Dong" uniqKey="Huang J" first="Jian-Dong" last="Huang">Jian-Dong Huang</name><name sortKey="Kung, Hsiang Fu" sort="Kung, Hsiang Fu" uniqKey="Kung H" first="Hsiang-Fu" last="Kung">Hsiang-Fu Kung</name><name sortKey="Lin, Marie C" sort="Lin, Marie C" uniqKey="Lin M" first="Marie C" last="Lin">Marie C. Lin</name><name sortKey="Lu, Lin Yu" sort="Lu, Lin Yu" uniqKey="Lu L" first="Lin-Yu" last="Lu">Lin-Yu Lu</name><name sortKey="Peiris, J S Malik" sort="Peiris, J S Malik" uniqKey="Peiris J" first="J S Malik" last="Peiris">J S Malik Peiris</name><name sortKey="Poon, Leo L M" sort="Poon, Leo L M" uniqKey="Poon L" first="Leo L M" last="Poon">Leo L M. Poon</name><name sortKey="Watt, Rory M" sort="Watt, Rory M" uniqKey="Watt R" first="Rory M" last="Watt">Rory M. Watt</name></noCountry><country name="République populaire de Chine"><noRegion><name sortKey="Tanner, Julian A" sort="Tanner, Julian A" uniqKey="Tanner J" first="Julian A" last="Tanner">Julian A. Tanner</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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