mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*
Identifieur interne : 000C54 ( Pmc/Curation ); précédent : 000C53; suivant : 000C55mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*
Auteurs : Hsiang-I. Liao ; C. Anders Olson [États-Unis] ; Seungmin Hwang ; Hongyu Deng ; Elaine Wong ; Ralph S. Baric ; Richard W. Roberts ; Ren SunSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2009.
Abstract
The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with
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DOI: 10.1074/jbc.M901547200
PubMed: 19364769
PubMed Central: 2719390
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></title><author><name sortKey="Liao, Hsiang I" sort="Liao, Hsiang I" uniqKey="Liao H" first="Hsiang-I." last="Liao">Hsiang-I. Liao</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Olson, C Anders" sort="Olson, C Anders" uniqKey="Olson C" first="C. Anders" last="Olson">C. Anders Olson</name><affiliation wicri:level="2"><nlm:aff id="aff3">Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125,</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena</wicri:cityArea></affiliation></author><author><name sortKey="Hwang, Seungmin" sort="Hwang, Seungmin" uniqKey="Hwang S" first="Seungmin" last="Hwang">Seungmin Hwang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Deng, Hongyu" sort="Deng, Hongyu" uniqKey="Deng H" first="Hongyu" last="Deng">Hongyu Deng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Wong, Elaine" sort="Wong, Elaine" uniqKey="Wong E" first="Elaine" last="Wong">Elaine Wong</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph S" sort="Baric, Ralph S" uniqKey="Baric R" first="Ralph S." last="Baric">Ralph S. Baric</name><affiliation><nlm:aff id="aff4"></nlm:aff></affiliation></author><author><name sortKey="Roberts, Richard W" sort="Roberts, Richard W" uniqKey="Roberts R" first="Richard W." last="Roberts">Richard W. Roberts</name><affiliation><nlm:aff id="aff5"></nlm:aff></affiliation></author><author><name sortKey="Sun, Ren" sort="Sun, Ren" uniqKey="Sun R" first="Ren" last="Sun">Ren Sun</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19364769</idno><idno type="pmc">2719390</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719390</idno><idno type="RBID">PMC:2719390</idno><idno type="doi">10.1074/jbc.M901547200</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000C54</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000C54</idno><idno type="wicri:Area/Pmc/Curation">000C54</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000C54</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></title><author><name sortKey="Liao, Hsiang I" sort="Liao, Hsiang I" uniqKey="Liao H" first="Hsiang-I." last="Liao">Hsiang-I. Liao</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Olson, C Anders" sort="Olson, C Anders" uniqKey="Olson C" first="C. Anders" last="Olson">C. Anders Olson</name><affiliation wicri:level="2"><nlm:aff id="aff3">Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125,</nlm:aff><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena</wicri:cityArea></affiliation></author><author><name sortKey="Hwang, Seungmin" sort="Hwang, Seungmin" uniqKey="Hwang S" first="Seungmin" last="Hwang">Seungmin Hwang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Deng, Hongyu" sort="Deng, Hongyu" uniqKey="Deng H" first="Hongyu" last="Deng">Hongyu Deng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Wong, Elaine" sort="Wong, Elaine" uniqKey="Wong E" first="Elaine" last="Wong">Elaine Wong</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph S" sort="Baric, Ralph S" uniqKey="Baric R" first="Ralph S." last="Baric">Ralph S. Baric</name><affiliation><nlm:aff id="aff4"></nlm:aff></affiliation></author><author><name sortKey="Roberts, Richard W" sort="Roberts, Richard W" uniqKey="Roberts R" first="Richard W." last="Roberts">Richard W. Roberts</name><affiliation><nlm:aff id="aff5"></nlm:aff></affiliation></author><author><name sortKey="Sun, Ren" sort="Sun, Ren" uniqKey="Sun R" first="Ren" last="Sun">Ren Sun</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"></nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with <italic>K<sub>d</sub></italic> = 1.7 n<sc>m</sc>. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly <italic>in vivo</italic> and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication.</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">J Biol Chem</journal-id><journal-id journal-id-type="hwp">jbc</journal-id><journal-id journal-id-type="pmc">jbc</journal-id><journal-id journal-id-type="publisher-id">JBC</journal-id><journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name><publisher-loc>9650 Rockville Pike, Bethesda, MD 20814, U.S.A.</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19364769</article-id><article-id pub-id-type="pmc">2719390</article-id><article-id pub-id-type="publisher-id">M901547200</article-id><article-id pub-id-type="doi">10.1074/jbc.M901547200</article-id><article-categories><subj-group subj-group-type="heading"><subject>Genomics, Proteomics, and Bioinformatics</subject></subj-group></article-categories><title-group><article-title>mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein<xref ref-type="fn" rid="FN1">*</xref><xref ref-type="fn" rid="FN2"><sup><inline-graphic xlink:href="sbox.jpg"></inline-graphic></sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Liao</surname><given-names>Hsiang-I.</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="author-notes" rid="FN3"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Olson</surname><given-names>C. Anders</given-names></name><xref ref-type="aff" rid="aff3"><sup>§</sup></xref><xref ref-type="author-notes" rid="FN3"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hwang</surname><given-names>Seungmin</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Deng</surname><given-names>Hongyu</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wong</surname><given-names>Elaine</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name><surname>Baric</surname><given-names>Ralph S.</given-names></name><xref ref-type="aff" rid="aff4"><sup>¶</sup></xref></contrib><contrib contrib-type="author"><name><surname>Roberts</surname><given-names>Richard W.</given-names></name><xref ref-type="aff" rid="aff5"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Ren</given-names></name><xref ref-type="aff" rid="aff1"><sup>‡</sup></xref><xref ref-type="aff" rid="aff2">**</xref><xref ref-type="corresp" rid="cor1"><sup>2</sup></xref></contrib><aff id="aff1">From the<label>‡</label>Department of Molecular and Medical Pharmacology and</aff><aff id="aff2">the<label>**</label>California Nano System Institute, UCLA, Los Angeles, California 90095,</aff><aff id="aff3"><label>§</label>Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125,</aff><aff id="aff4">the<label>¶</label>Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina 27599, and</aff><aff id="aff5">the<label>‖</label>Department of Chemistry, Chemical Engineering, and Biology, University of Southern California, Los Angeles, California 90089-1211</aff></contrib-group><author-notes><corresp id="cor1"><label>2</label>To whom correspondence should be addressed:
<addr-line>650 Charles E. Young Dr. South, CHS 23-120, UCLA, Los Angeles, CA 90095.</addr-line> Fax:
<fax>310-825-6267</fax>; E-mail: <email>rsun@mednet.ucla.edu</email>.</corresp><fn fn-type="equal" id="FN3"><label>1</label><p>Both authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="ppub"><day>26</day><month>6</month><year>2009</year></pub-date><pub-date pub-type="epub"><day>13</day><month>4</month><year>2009</year></pub-date><volume>284</volume><issue>26</issue><fpage>17512</fpage><lpage>17520</lpage><history><date date-type="received"><day>6</day><month>3</month><year>2009</year></date></history><permissions><copyright-statement>© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zbc02609017512.pdf"></self-uri><abstract><p>The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with <italic>K<sub>d</sub></italic> = 1.7 n<sc>m</sc>. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly <italic>in vivo</italic> and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication.</p></abstract><funding-group><award-group><funding-source id="CS100">National Institutes of Health</funding-source><award-id rid="CS100">RO1 GM60416</award-id></award-group></funding-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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