Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase
Identifieur interne : 001589 ( Pmc/Checkpoint ); précédent : 001588; suivant : 001590Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase
Auteurs : Konstantin A. Ivanov ; Volker Thiel ; Jessika C. Dobbe ; Yvonne Van Der Meer ; Eric J. Snijder ; John ZiebuhrSource :
- Journal of Virology [ 0022-538X ] ; 2004.
Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
Url:
DOI: 10.1128/JVI.78.11.5619-5632.2004
PubMed: 15140959
PubMed Central: 415832
Affiliations:
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<front><div type="abstract" xml:lang="en"><p>Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.</p>
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<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Virol</journal-id>
<journal-id journal-id-type="publisher-id">jvi</journal-id>
<journal-title>Journal of Virology</journal-title>
<issn pub-type="ppub">0022-538X</issn>
<issn pub-type="epub">1098-5514</issn>
<publisher><publisher-name>American Society for Microbiology</publisher-name>
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<article-id pub-id-type="pmc">415832</article-id>
<article-id pub-id-type="publisher-id">2551-03</article-id>
<article-id pub-id-type="doi">10.1128/JVI.78.11.5619-5632.2004</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Replication</subject>
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</article-categories>
<title-group><article-title>Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Ivanov</surname>
<given-names>Konstantin A.</given-names>
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<xref ref-type="aff" rid="aff1">1</xref>
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<contrib contrib-type="author"><name><surname>Thiel</surname>
<given-names>Volker</given-names>
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<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="fn" rid="fn1">†</xref>
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<contrib contrib-type="author"><name><surname>Dobbe</surname>
<given-names>Jessika C.</given-names>
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<contrib contrib-type="author"><name><surname>van der Meer</surname>
<given-names>Yvonne</given-names>
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<xref ref-type="aff" rid="aff1">2</xref>
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<contrib contrib-type="author"><name><surname>Snijder</surname>
<given-names>Eric J.</given-names>
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<contrib contrib-type="author"><name><surname>Ziebuhr</surname>
<given-names>John</given-names>
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<aff id="aff1">Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany,<label>1</label>
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands<label>2</label>
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<author-notes><fn id="cor1"><label>*</label>
<p>Corresponding author. Mailing address: Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany. Phone: 49-931-20149928. Fax: 49-931-20149553. E-mail: <email>j.ziebuhr@mail.uni-wuerzburg.de</email>
.</p>
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<fn id="fn1"><label>†</label>
<p>Present address: Research Department, Cantonal Hospital, St. Gallen, Switzerland.</p>
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<pub-date pub-type="ppub"><month>6</month>
<year>2004</year>
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<volume>78</volume>
<issue>11</issue>
<fpage>5619</fpage>
<lpage>5632</lpage>
<history><date date-type="received"><day>4</day>
<month>12</month>
<year>2003</year>
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<date date-type="accepted"><day>26</day>
<month>1</month>
<year>2004</year>
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<copyright-statement>Copyright © 2004, American Society for Microbiology</copyright-statement>
<copyright-year>2004</copyright-year>
<abstract><p>Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5′-to-3′ direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5′-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5′ cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5′-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.</p>
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<tree><noCountry><name sortKey="Dobbe, Jessika C" sort="Dobbe, Jessika C" uniqKey="Dobbe J" first="Jessika C." last="Dobbe">Jessika C. Dobbe</name>
<name sortKey="Ivanov, Konstantin A" sort="Ivanov, Konstantin A" uniqKey="Ivanov K" first="Konstantin A." last="Ivanov">Konstantin A. Ivanov</name>
<name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name>
<name sortKey="Thiel, Volker" sort="Thiel, Volker" uniqKey="Thiel V" first="Volker" last="Thiel">Volker Thiel</name>
<name sortKey="Van Der Meer, Yvonne" sort="Van Der Meer, Yvonne" uniqKey="Van Der Meer Y" first="Yvonne" last="Van Der Meer">Yvonne Van Der Meer</name>
<name sortKey="Ziebuhr, John" sort="Ziebuhr, John" uniqKey="Ziebuhr J" first="John" last="Ziebuhr">John Ziebuhr</name>
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