One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities
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Auteurs : Lorenzo Subissi [France] ; Clara C. Posthuma [Pays-Bas] ; Axelle Collet [France] ; Jessika C. Zevenhoven-Dobbe [Pays-Bas] ; Alexander E. Gorbalenya [Pays-Bas, Russie] ; Etienne Decroly [France] ; Eric J. Snijder [Pays-Bas] ; Bruno Canard [France] ; Isabelle Imbert [France]Source :
- Proceedings of the National Academy of Sciences of the United States of America [ 0027-8424 ] ; 2014.
Abstract
The 2003 severe acute respiratory syndrome (SARS) epidemic and recent emergence of Middle East respiratory syndrome highlight the potential lethality of zoonotic coronavirus infections in humans. No specific antiviral treatment options are available. Coronaviruses possess the largest known RNA virus genomes and encode a complex replication machinery consisting of 16 viral nonstructural proteins (nsps). Our study reveals that the SARS-coronavirus RNA polymerase (nsp12) needs to associate with nsp7 and nsp8 to activate its capability to replicate long RNA. Moreover, this complex associates with nsp14, the proofreading subunit required to safeguard coronavirus replication fidelity. Our study thus defines the core of an RNA-synthesizing machinery that is unique in the RNA virus world and includes several key targets for antiviral drug development.
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DOI: 10.1073/pnas.1323705111
PubMed: 25197083
PubMed Central: 4169972
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Snijder</name><affiliation wicri:level="1"><nlm:aff wicri:cut="; and" id="aff2">Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Canard, Bruno" sort="Canard, Bruno" uniqKey="Canard B" first="Bruno" last="Canard">Bruno Canard</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Imbert, Isabelle" sort="Imbert, Isabelle" uniqKey="Imbert I" first="Isabelle" last="Imbert">Isabelle Imbert</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25197083</idno><idno type="pmc">4169972</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169972</idno><idno type="RBID">PMC:4169972</idno><idno type="doi">10.1073/pnas.1323705111</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000D17</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D17</idno><idno type="wicri:Area/Pmc/Curation">000D17</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000D17</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities</title><author><name sortKey="Subissi, Lorenzo" sort="Subissi, Lorenzo" uniqKey="Subissi L" first="Lorenzo" last="Subissi">Lorenzo Subissi</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Posthuma, Clara C" sort="Posthuma, Clara C" uniqKey="Posthuma C" first="Clara C." last="Posthuma">Clara C. Posthuma</name><affiliation wicri:level="1"><nlm:aff wicri:cut="; and" id="aff2">Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Collet, Axelle" sort="Collet, Axelle" uniqKey="Collet A" first="Axelle" last="Collet">Axelle Collet</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Zevenhoven Dobbe, Jessika C" sort="Zevenhoven Dobbe, Jessika C" uniqKey="Zevenhoven Dobbe J" first="Jessika C." last="Zevenhoven-Dobbe">Jessika C. Zevenhoven-Dobbe</name><affiliation wicri:level="1"><nlm:aff wicri:cut="; and" id="aff2">Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E." last="Gorbalenya">Alexander E. Gorbalenya</name><affiliation wicri:level="1"><nlm:aff wicri:cut="; and" id="aff2">Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff3">Faculty of Bioengineering and Bioinformatics,<institution>Lomonosov Moscow State University</institution>, Moscow 119899,<country>Russia</country></nlm:aff><country xml:lang="fr">Russie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Decroly, Etienne" sort="Decroly, Etienne" uniqKey="Decroly E" first="Etienne" last="Decroly">Etienne Decroly</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name><affiliation wicri:level="1"><nlm:aff wicri:cut="; and" id="aff2">Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Canard, Bruno" sort="Canard, Bruno" uniqKey="Canard B" first="Bruno" last="Canard">Bruno Canard</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Imbert, Isabelle" sort="Imbert, Isabelle" uniqKey="Imbert I" first="Isabelle" last="Imbert">Isabelle Imbert</name><affiliation wicri:level="1"><nlm:aff id="aff1">Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Proceedings of the National Academy of Sciences of the United States of America</title><idno type="ISSN">0027-8424</idno><idno type="eISSN">1091-6490</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Significance</title><p>The 2003 severe acute respiratory syndrome (SARS) epidemic and recent emergence of Middle East respiratory syndrome highlight the potential lethality of zoonotic coronavirus infections in humans. No specific antiviral treatment options are available. Coronaviruses possess the largest known RNA virus genomes and encode a complex replication machinery consisting of 16 viral nonstructural proteins (nsps). Our study reveals that the SARS-coronavirus RNA polymerase (nsp12) needs to associate with nsp7 and nsp8 to activate its capability to replicate long RNA. Moreover, this complex associates with nsp14, the proofreading subunit required to safeguard coronavirus replication fidelity. Our study thus defines the core of an RNA-synthesizing machinery that is unique in the RNA virus world and includes several key targets for antiviral drug development.</p></div></front></TEI><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">Proc Natl Acad Sci U S A</journal-id><journal-id journal-id-type="iso-abbrev">Proc. Natl. Acad. Sci. U.S.A</journal-id><journal-id journal-id-type="hwp">pnas</journal-id><journal-id journal-id-type="pmc">pnas</journal-id><journal-id journal-id-type="publisher-id">PNAS</journal-id><journal-title-group><journal-title>Proceedings of the National Academy of Sciences of the United States of America</journal-title></journal-title-group><issn pub-type="ppub">0027-8424</issn><issn pub-type="epub">1091-6490</issn><publisher><publisher-name>National Academy of Sciences</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25197083</article-id><article-id pub-id-type="pmc">4169972</article-id><article-id pub-id-type="publisher-id">201323705</article-id><article-id pub-id-type="doi">10.1073/pnas.1323705111</article-id><article-categories><subj-group subj-group-type="heading"><subject>PNAS Plus</subject></subj-group><subj-group subj-group-type="heading"><subject>Biological Sciences</subject><subj-group><subject>Microbiology</subject></subj-group></subj-group><series-title>PNAS Plus</series-title></article-categories><title-group><article-title>One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities</article-title><alt-title alt-title-type="short">Processive SARS-CoV RNA polymerase complex</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Subissi</surname><given-names>Lorenzo</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Posthuma</surname><given-names>Clara C.</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Collet</surname><given-names>Axelle</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zevenhoven-Dobbe</surname><given-names>Jessika C.</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Gorbalenya</surname><given-names>Alexander E.</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref><xref ref-type="aff" rid="aff3"><sup>c</sup></xref></contrib><contrib contrib-type="author"><name><surname>Decroly</surname><given-names>Etienne</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Snijder</surname><given-names>Eric J.</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref></contrib><contrib contrib-type="author"><name><surname>Canard</surname><given-names>Bruno</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref><xref ref-type="corresp" rid="cor1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Imbert</surname><given-names>Isabelle</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref><xref ref-type="corresp" rid="cor1"><sup>1</sup></xref></contrib><aff id="aff1"><sup>a</sup>Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257,<institution>Aix-Marseille Université</institution>, 13288 Marseille,<country>France</country>;</aff><aff id="aff2"><sup>b</sup>Molecular Virology Laboratory, Department of Medical Microbiology,<institution>Leiden University Medical Center</institution>, 2300RC, Leiden,<country>The Netherlands</country>; and</aff><aff id="aff3"><sup>c</sup>Faculty of Bioengineering and Bioinformatics,<institution>Lomonosov Moscow State University</institution>, Moscow 119899,<country>Russia</country></aff></contrib-group><author-notes><corresp id="cor1"><sup>1</sup>To whom correspondence may be addressed. Email: <email>isabelle.imbert@afmb.univ-mrs.fr</email> or <email>bruno.canard@afmb.univ-mrs.fr</email>.</corresp><fn fn-type="edited-by"><p>Edited by Paul Ahlquist, University of Wisconsin, Madison, WI, and approved August 5, 2014 (received for review December 19, 2013)</p></fn><fn fn-type="con"><p>Author contributions: L.S., C.C.P., A.E.G., E.D., E.J.S., B.C., and I.I. designed research; L.S., C.C.P., A.C., J.C.Z.-D., E.J.S., B.C., and I.I. performed research; A.E.G. contributed new reagents/analytic tools; L.S., C.C.P., A.E.G., E.D., E.J.S., B.C., and I.I. analyzed data; and L.S., C.C.P., E.D., E.J.S., B.C., and I.I. wrote the paper.</p></fn><fn fn-type="conflict"><p>The authors declare no conflict of interest.</p></fn></author-notes><pub-date pub-type="ppub"><day>16</day><month>9</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>2</day><month>9</month><year>2014</year></pub-date><volume>111</volume><issue>37</issue><fpage>E3900</fpage><lpage>E3909</lpage><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="pnas.201323705.pdf"></self-uri><abstract abstract-type="executive-summary"><title>Significance</title><p>The 2003 severe acute respiratory syndrome (SARS) epidemic and recent emergence of Middle East respiratory syndrome highlight the potential lethality of zoonotic coronavirus infections in humans. No specific antiviral treatment options are available. Coronaviruses possess the largest known RNA virus genomes and encode a complex replication machinery consisting of 16 viral nonstructural proteins (nsps). Our study reveals that the SARS-coronavirus RNA polymerase (nsp12) needs to associate with nsp7 and nsp8 to activate its capability to replicate long RNA. Moreover, this complex associates with nsp14, the proofreading subunit required to safeguard coronavirus replication fidelity. Our study thus defines the core of an RNA-synthesizing machinery that is unique in the RNA virus world and includes several key targets for antiviral drug development.</p></abstract><abstract><p>In addition to members causing milder human infections, the <italic>Coronaviridae</italic> family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.</p></abstract><kwd-group><kwd>replicative complex reconstitution</kwd><kwd>processivity factor</kwd></kwd-group><counts><page-count count="10"></page-count></counts></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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