mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*
Identifieur interne : 000C54 ( Pmc/Corpus ); 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 ; 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
Url:
DOI: 10.1074/jbc.M901547200
PubMed: 19364769
PubMed Central: 2719390
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PMC:2719390Le document en format XML
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<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>
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<author><name sortKey="Liao, Hsiang I" sort="Liao, Hsiang I" uniqKey="Liao H" first="Hsiang-I." last="Liao">Hsiang-I. Liao</name>
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<author><name sortKey="Olson, C Anders" sort="Olson, C Anders" uniqKey="Olson C" first="C. Anders" last="Olson">C. Anders Olson</name>
<affiliation><nlm:aff id="aff3">Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125,</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Hwang, Seungmin" sort="Hwang, Seungmin" uniqKey="Hwang S" first="Seungmin" last="Hwang">Seungmin Hwang</name>
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<author><name sortKey="Deng, Hongyu" sort="Deng, Hongyu" uniqKey="Deng H" first="Hongyu" last="Deng">Hongyu Deng</name>
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<author><name sortKey="Wong, Elaine" sort="Wong, Elaine" uniqKey="Wong E" first="Elaine" last="Wong">Elaine Wong</name>
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<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>
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<author><name sortKey="Roberts, Richard W" sort="Roberts, Richard W" uniqKey="Roberts R" first="Richard W." last="Roberts">Richard W. Roberts</name>
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<author><name sortKey="Sun, Ren" sort="Sun, Ren" uniqKey="Sun R" first="Ren" last="Sun">Ren Sun</name>
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<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>
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<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>
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