A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation
Identifieur interne : 000934 ( Pmc/Curation ); précédent : 000933; suivant : 000935A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation
Auteurs : Meike Dittmann [États-Unis] ; Hans-Heinrich Hoffmann [États-Unis] ; Margaret A. Scull [États-Unis] ; Rachel H. Gilmore [États-Unis] ; Kierstin L. Bell [États-Unis] ; Michael Ciancanelli [États-Unis] ; Sam J. Wilson [États-Unis, Royaume-Uni] ; Stefania Crotta [Royaume-Uni] ; Yingpu Yu [États-Unis] ; Brenna Flatley [États-Unis] ; Jing W. Xiao [États-Unis] ; Jean-Laurent Casanova [États-Unis, France] ; Andreas Wack [Royaume-Uni] ; Paul D. Bieniasz [États-Unis] ; Charles M. Rice [États-Unis]Source :
- Cell [ 0092-8674 ] ; 2015.
Abstract
Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene
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DOI: 10.1016/j.cell.2015.01.040
PubMed: 25679759
PubMed Central: 4328142
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Bieniasz</name><affiliation wicri:level="1"><nlm:aff id="aff3">Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016</wicri:regionArea></affiliation></author><author><name sortKey="Rice, Charles M" sort="Rice, Charles M" uniqKey="Rice C" first="Charles M." last="Rice">Charles M. Rice</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25679759</idno><idno type="pmc">4328142</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4328142</idno><idno type="RBID">PMC:4328142</idno><idno type="doi">10.1016/j.cell.2015.01.040</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000934</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000934</idno><idno type="wicri:Area/Pmc/Curation">000934</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000934</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation</title><author><name sortKey="Dittmann, Meike" sort="Dittmann, Meike" uniqKey="Dittmann M" first="Meike" last="Dittmann">Meike Dittmann</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Hoffmann, Hans Heinrich" sort="Hoffmann, Hans Heinrich" uniqKey="Hoffmann H" first="Hans-Heinrich" last="Hoffmann">Hans-Heinrich Hoffmann</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Scull, Margaret A" sort="Scull, Margaret A" uniqKey="Scull M" first="Margaret A." last="Scull">Margaret A. Scull</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Gilmore, Rachel H" sort="Gilmore, Rachel H" uniqKey="Gilmore R" first="Rachel H." last="Gilmore">Rachel H. Gilmore</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Bell, Kierstin L" sort="Bell, Kierstin L" uniqKey="Bell K" first="Kierstin L." last="Bell">Kierstin L. Bell</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Ciancanelli, Michael" sort="Ciancanelli, Michael" uniqKey="Ciancanelli M" first="Michael" last="Ciancanelli">Michael Ciancanelli</name><affiliation wicri:level="1"><nlm:aff id="aff2">St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Wilson, Sam J" sort="Wilson, Sam J" uniqKey="Wilson S" first="Sam J." last="Wilson">Sam J. Wilson</name><affiliation wicri:level="1"><nlm:aff id="aff3">Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016</wicri:regionArea></affiliation><affiliation wicri:level="4"><nlm:aff id="aff4">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK</nlm:aff><orgName type="university">Université de Glasgow</orgName><country>Royaume-Uni</country><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Crotta, Stefania" sort="Crotta, Stefania" uniqKey="Crotta S" first="Stefania" last="Crotta">Stefania Crotta</name><affiliation wicri:level="1"><nlm:aff id="aff5">Division of Immunoregulation, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Division of Immunoregulation, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA</wicri:regionArea></affiliation></author><author><name sortKey="Yu, Yingpu" sort="Yu, Yingpu" uniqKey="Yu Y" first="Yingpu" last="Yu">Yingpu Yu</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Flatley, Brenna" sort="Flatley, Brenna" uniqKey="Flatley B" first="Brenna" last="Flatley">Brenna Flatley</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Xiao, Jing W" sort="Xiao, Jing W" uniqKey="Xiao J" first="Jing W." last="Xiao">Jing W. Xiao</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author><author><name sortKey="Casanova, Jean Laurent" sort="Casanova, Jean Laurent" uniqKey="Casanova J" first="Jean-Laurent" last="Casanova">Jean-Laurent Casanova</name><affiliation wicri:level="1"><nlm:aff id="aff2">St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff6">Howard Hughes Medical Institute, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Howard Hughes Medical Institute, NY 10065</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff7">Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Imagine Institute, Inserm, 75015 Paris, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Imagine Institute, Inserm, 75015 Paris</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff8">Paris Descartes University, 75015 Paris, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Paris Descartes University, 75015 Paris</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="aff9">Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, 75015 Paris, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, 75015 Paris</wicri:regionArea></affiliation></author><author><name sortKey="Wack, Andreas" sort="Wack, Andreas" uniqKey="Wack A" first="Andreas" last="Wack">Andreas Wack</name><affiliation wicri:level="1"><nlm:aff id="aff5">Division of Immunoregulation, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Division of Immunoregulation, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA</wicri:regionArea></affiliation></author><author><name sortKey="Bieniasz, Paul D" sort="Bieniasz, Paul D" uniqKey="Bieniasz P" first="Paul D." last="Bieniasz">Paul D. Bieniasz</name><affiliation wicri:level="1"><nlm:aff id="aff3">Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016</wicri:regionArea></affiliation></author><author><name sortKey="Rice, Charles M" sort="Rice, Charles M" uniqKey="Rice C" first="Charles M." last="Rice">Charles M. Rice</name><affiliation wicri:level="1"><nlm:aff id="aff1">Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065</wicri:regionArea></affiliation></author></analytic><series><title level="j">Cell</title><idno type="ISSN">0092-8674</idno><idno type="eISSN">1097-4172</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene <italic>SERPINE1</italic>, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human <italic>SERPINE1</italic>, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Beppu, Y" uniqKey="Beppu Y">Y. Beppu</name></author><author><name sortKey="Imamura, Y" uniqKey="Imamura Y">Y. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell</journal-id><journal-id journal-id-type="iso-abbrev">Cell</journal-id><journal-title-group><journal-title>Cell</journal-title></journal-title-group><issn pub-type="ppub">0092-8674</issn><issn pub-type="epub">1097-4172</issn><publisher><publisher-name>Cell Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25679759</article-id><article-id pub-id-type="pmc">4328142</article-id><article-id pub-id-type="publisher-id">S0092-8674(15)00118-X</article-id><article-id pub-id-type="doi">10.1016/j.cell.2015.01.040</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Dittmann</surname><given-names>Meike</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Hoffmann</surname><given-names>Hans-Heinrich</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Scull</surname><given-names>Margaret A.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Gilmore</surname><given-names>Rachel H.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Bell</surname><given-names>Kierstin L.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Ciancanelli</surname><given-names>Michael</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Wilson</surname><given-names>Sam J.</given-names></name><xref rid="aff3" ref-type="aff">3</xref><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Crotta</surname><given-names>Stefania</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Yingpu</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Flatley</surname><given-names>Brenna</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Xiao</surname><given-names>Jing W.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Casanova</surname><given-names>Jean-Laurent</given-names></name><xref rid="aff2" ref-type="aff">2</xref><xref rid="aff6" ref-type="aff">6</xref><xref rid="aff7" ref-type="aff">7</xref><xref rid="aff8" ref-type="aff">8</xref><xref rid="aff9" ref-type="aff">9</xref></contrib><contrib contrib-type="author"><name><surname>Wack</surname><given-names>Andreas</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Bieniasz</surname><given-names>Paul D.</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author"><name><surname>Rice</surname><given-names>Charles M.</given-names></name><email>ricec@rockefeller.edu</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</aff><aff id="aff2"><label>2</label>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA</aff><aff id="aff3"><label>3</label>Howard Hughes Medical Institute, Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY 10016, USA</aff><aff id="aff4"><label>4</label>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK</aff><aff id="aff5"><label>5</label>Division of Immunoregulation, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK</aff><aff id="aff6"><label>6</label>Howard Hughes Medical Institute, NY 10065, USA</aff><aff id="aff7"><label>7</label>Laboratory of Human Genetics of Infectious Diseases, Necker Branch, Imagine Institute, Inserm, 75015 Paris, France</aff><aff id="aff8"><label>8</label>Paris Descartes University, 75015 Paris, France</aff><aff id="aff9"><label>9</label>Pediatric Hematology-Immunology Unit, Necker Hospital for Sick Children, 75015 Paris, France</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>ricec@rockefeller.edu</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>12</day><month>2</month><year>2015</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>12</day><month>2</month><year>2015</year></pub-date><volume>160</volume><issue>4</issue><fpage>631</fpage><lpage>643</lpage><history><date date-type="received"><day>19</day><month>9</month><year>2014</year></date><date date-type="rev-recd"><day>22</day><month>10</month><year>2014</year></date><date date-type="accepted"><day>13</day><month>1</month><year>2015</year></date></history><permissions><copyright-statement>© 2015 The Authors. Published by Elsevier Inc.</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license license-type="CC BY" xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</license-p></license></permissions><abstract><title>Summary</title><p>Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene <italic>SERPINE1</italic>, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human <italic>SERPINE1</italic>, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.</p></abstract><abstract abstract-type="graphical"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights"><title>Highlights</title><p><list list-type="simple"><list-item id="u0010"><label>•</label><p><italic>SERPINE1</italic>/PAI-1 was identified as an unconventional ISG that acts extracellularly</p></list-item><list-item id="u0015"><label>•</label><p>PAI-1 inhibits influenza A virus (IAV) spread by inhibiting glycoprotein cleavage</p></list-item><list-item id="u0020"><label>•</label><p>Endogenous PAI-1 blocks IAV spread in human and murine cells, ex vivo and in vivo</p></list-item><list-item id="u0025"><label>•</label><p>PAI-1 potentially inhibits other viruses requiring extracellular maturation</p></list-item></list></p></abstract><abstract abstract-type="teaser"><p>Plasminogen activator inhibitor (PAI-1) blocks surface glycoprotein maturation of influenza A virus, thus reducing virus spread in the airways and revealing that the innate immune system, driven by type I IFN, uses modulation of the extracellular environment to inhibit viruses.</p></abstract></article-meta></front><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>High-Throughput Microscopy Screens for Inhibitors of IAV Spread</p><p>(A) Screening workflow. Shown are hypothetical effects of expressing inhibitory (antiviral) or non-inhibitory ISGs on single or multiple rounds of virus replication. Red, transduced cells; green, infected cells; blue, DAPI-stained nuclei.</p><p>(B) Effect of 401 single ISGs on IAV spread. ISGs inhibiting more than 2-fold SD in two independent screens are shown in red. Spread ratio, the ratio of infected cells at 24/8 hpi. A positive control for inhibition is α-HA antibody.</p><p>(C) Confirmation assays for selected ISGs on A549 cells or primary NHBE cells. Data are represented as mean ± SEM from n = 6 values in two independent experiments for A549, and n = 3 for NHBE cells.</p><p>(D) <italic>SERPINE1</italic>(PAI-1)-mediated inhibition of A/Vietnam/1203/2004(HALo), A/California/04/2009, and A/sw/Texas/4199-2/1998 in A549 cells. Empty vector, negative control; <italic>IFITM3</italic> and <italic>BST2</italic> (tetherin), positive controls. Data are represented as mean ± SEM from n = 4 independent experiments. One-way ANOVA and Dunn’s multiple comparison test versus “empty.”</p><p>(E) ISG-expressing A549 cells were infected with IAV WSN/33 at MOI 0.01, and virus titers were measured by plaque assay on MDCK cells. Data are represented as mean ± SEM from n = 4 independent experiments.</p><p>See also <xref rid="figs1" ref-type="fig">Figure S1</xref>.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p><italic>SERPINE1</italic> Gene Expression Profiles and the Role of Extracellular PAI-1 Protein for IAV Inhibition</p><p>(A–C) A549 cells were infected with IAV WSN/33, treated with IFN-β, or mock-treated. (A) <italic>SERPINE1</italic> mRNA expression was normalized relative to housekeeping gene <italic>RPS-11</italic>. Fold increase over pre-treatment control levels is shown. Accumulated total (B) intracellular and (C) extracellular protein levels of PAI-1 were determined by ELISA. Data are represented as mean ± SEM from n = 3 experiments.</p><p>(D) HAEC infected with IAV WSN/33 or mock treated. Accumulated PAI-1 protein in repeated apical washes was determined by ELISA. Data are shown as mean ± SEM from n = 3 replicates.</p><p>(E) A549 cells were infected with IAV WSN/33 in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM at 48 hpi. The number of infected cells was normalized to buffer (1) or IgG (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests.</p><p>(F) HAECs were infected with IAV WSN/33 in the presence of either rPAI (1) or α-PAI-1 (2) and buffer (1) or IgG (2). Progeny virus was collected from apical washes, and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by plaque assay. Data are shown as mean ± SEM from n = 3 replicates.</p><p>See also <xref rid="figs2" ref-type="fig">Figure S2</xref>.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>Protease Targets of PAI-1</p><p>(A–D) Representative gel shift assays to show complexes of recombinant PAI-1 (rPAI-1) and indicated proteases. 1 μg of rPAI-1 was combined with increasing amounts of protease, and the mixture was separated on SDS gels followed by silver staining. Where indicated, triplaxtinin was used to inhibit PAI-1 activity.</p><p>(E–G) HTM spread assay in the presence of PAI-1 wild-type (<italic>SERPINE1</italic>), loss-of-function mutant (<italic>SERPINE1</italic><sup>∗</sup>), and controls for HPIV3-GFP (E), IAV WSN/33 (F), or SeV-GFP (G). Data are shown as mean ± SEM from at least n = 6 replicates from 2 independent experiments. One-way ANOVA and Kruskal-Wallis post-test.</p><p>See also <xref rid="figs4" ref-type="fig">Figure S4</xref>.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>Effect of PAI-1 on Maturation of IAV HA</p><p>(A, D, and E) BHK cells were transfected in serum-free MEM to express HA of different origins. Whole-cell lysates were analyzed by western blot. HA cleavage products HA1 and HA2 are indicated, and relative band intensities are shown at the bottom of the gel.</p><p>(A) HA of A/Puerto Rico/8/34 origin. Shown are cells treated with TPCK-trypsin, TPCK-trypsin and rPAI-1, or buffer.</p><p>(B) BHK cells were transfected to express GFP as negative control, PAI-1 wild-type (<italic>SER1</italic>), or loss-of-function mutant (<italic>SER</italic><sup>∗</sup>) and then challenged with IAV Puerto Rico/8/1934 in the presence of TPCK-trypsin. Supernatants were harvested and assayed for infectivity by focus forming assay on MDCK cells and for viral RNA by qRT-PCR. Plaque assays were performed in duplicate with or without additional post-harvest incubation with TPCK-trypsin. Specific infectivity was determined by calculating the ratio of FFU to vRNA copies, and values were normalized to GFP control. Data are shown as mean ± SEM from n = 6 replicates from 2 independent experiments.</p><p>(C) BHK cells were transfected to co-express GFP as negative control, PAI-1 wild-type (<italic>SER</italic>), or loss-of-function mutant (<italic>SER</italic><sup>∗</sup>), and TMPRSS2 or HAT as bait protease. Cells were then challenged, and virus infectivity was analyzed as described in (B).</p><p>(D) HA cleavage assay with H1 of A/Puerto Rico/8/34 or H2 of A/Japan/305/57 origin in the presence of protease TMPRSS2 and wild-type (<italic>SER</italic>) or loss-of-function (<italic>SER</italic><sup>∗</sup>) PAI-1.</p><p>(E) HA cleavage assay with H5 of A/Vietnam/1203/2004 origin and wild-type (<italic>SER</italic>) PAI-1, or GFP as a negative control.</p><p>(F) HA cleavage assay on HAEC infected with IAV WSN/33 in the presence of either rPAI (1) or α-PAI-1 (2) and buffer (1) or IgG (2). Progeny virus particles from apical washes at 24 hpi were analyzed.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p>Role of <italic>Serpine1</italic> in Lethal IAV Infection in Mice</p><p>(A–F) Wild-type (B6) or <italic>Serpine1</italic><sup>−/−</sup> mice were infected intranasally with 36 LD<sub>50</sub> of IAV Puerto Rico/8/34. Statistical significance was determined by individual t tests (D, F, G, and I) or log rank test (E and H).</p><p>(A) Representative images of infected lungs 5 dpi.</p><p>(B and C) Homogenates of infected mouse lungs were assayed for <italic>Serpine1</italic> mRNA levels by qRT-PCR (B) and mPAI-1 protein levels by ELISA (C). Data are shown as mean ± SEM from n = 5 mice per group.</p><p>(D and E) Weight loss (D) and survival (E) of infected mice. Data are shown as mean ± SEM from n = 25 infected B6 or <italic>Serpine1</italic><sup>−/−</sup> mice, and n = 3 respective PBS control animals.</p><p>(F) Homogenates of infected lungs were assayed for IAV titers by focus forming assay on MDCK cells. Data are shown as mean ± SEM from n = 17 B6 and 11 <italic>Serpine1</italic><sup>−/−</sup> mice per day.</p><p>(G–I) Wild-type (B6) or <italic>Serpine1</italic><sup>−/−</sup> mice were infected intranasally with 36 LD<sub>50</sub> of IAV WSN/33 and monitored for weight loss (G) and survival (H). Data are shown as mean ± SEM from n = 32 infected B6 or <italic>Serpine1</italic><sup>−/−</sup> mice, and n = 3 respective PBS control animals. IAV lung titers (I) shown as mean ± SEM from n = 9 B6 or <italic>Serpine1</italic><sup>−/−</sup> mice per day.</p><p>(J) MTECs cultured from B6 or <italic>Serpine1</italic><sup>−/−</sup> mice were infected with A/X-31(H3N2) at MOI = 10<sup>−6</sup>. vRNA was quantified from lysed MTEC by qRT-PCR. In parallel, infectious virus titers were determined by TCID<sub>50</sub> assay on MDCK cells. Data are shown as mean ± SEM from n = 3 B6 or <italic>Serpine1</italic><sup>−/−</sup> MTECs. Statistical significance was determined by two-way ANOVA.</p><p>See also <xref rid="figs5" ref-type="fig">Figure S5</xref>.</p></caption><graphic xlink:href="gr5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p>Susceptibility of PAI-1-Deficient Human Fibroblasts to IAV</p><p>(A–H) SV-40-immortalized human fibroblasts from different donors, wild-type (T/T 1-3), heterozygous (T/A 4 and 5), or homozygous (A/A 6) for <italic>SERPINE1</italic> SNP rs6092 were infected with IAV WSN/33 (A–D, H, and I) or HPIV3-GFP (E–G).</p><p>(A) Representative images from single wells infected with IAV WSN/33 at 48 hpi.</p><p>(B) IAV spread shown as the ratio between number of infected cells at 12/48 hpi.</p><p>(C) Percent of infected fibroblasts after one round of replication. Data in (A) and (B) are represented as mean ± SEM from n = 4 independent experiments, each in triplicate.</p><p>(D) IAV WSN/33 growth on fibroblasts. PFU in the supernatants were determined by plaque assay on MDCK cells. Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance was determined by individual t test, comparing the pooled SNP-carrying cells with the pooled control cells TT2 and TT3 for every time point.</p><p>(E) HPIV3-GFP spread shown as the ratio between number of infected cells at 48 and 24 hpi.</p><p>(F) Percent of infected fibroblasts after one round of replication. Data in (E) and (F) are represented as mean ± SEM from n = 2 independent experiments, each in triplicate.</p><p>(G) HPIV3-GFP growth on fibroblasts. TCID<sub>50</sub> in supernatants were determined on LLC-MK2 cells.</p><p>(H) Extracellular PAI-1 levels from IAV WSN/33-infected fibroblasts during spread assay at 48 hpi. Data are shown as mean ± SEM from total n = 8 replicates from n = 4 experiments.</p><p>(I) Rescue of virus inhibition on selected human fibroblast lines by adding rPAI-1 to the cultures during IAV WSN/33 infection. Maximum addition of rPAI-1 (black bars) was 500 ng, minimum addition (light gray bars) 125 ng. Data are represented as mean ± SEM from n = 4 independent experiments, each in quadruplicate. One-way ANOVA and Dunnett’s multiple comparison post-test against T/T 1- values of the same condition.</p></caption><graphic xlink:href="gr6"></graphic></fig><fig id="figs1"><label>Figure S1</label><caption><p>High-Throughput Microscopy Screens for Inhibitors of IAV Spread, Related to <xref rid="fig1" ref-type="fig">Figure 1</xref></p><p>(A) Example of automated cell scoring from the HTM screen. Images show one representative out of 48 views per 96-well; original images from individual channels on the left (blue, DAPI-stained nuclei; red, transduced cells; green, NP-positive cells), and segmented images on the right (gray, nuclei; red, transduced cells; green, NP-positive cells).</p><p>(B) Establishing the spread ratio as a stable measure of spread over a large range of transduction efficiencies. A549 cells were transduced with a dilution series of <italic>BST2-</italic>expressing lentivirus, or a high dose of empty vector, the latter yielding 90% of transduced cells. A/WSN/33 spread was determined as described in <xref rid="fig1" ref-type="fig">Figure 1</xref>A.</p><p>(C) Establishing α-HA antibody to block IAV spread. A549 cells were infected with IAV WSN/33 for 24h, in the presence or absence of an α-HA antibody serial dilution, then fixed and stained for IAV NP. Shown are results from automated quantification, and representative images of two wells with the minimum inhibitory concentration, 0.125 μg/ml, or without α-HA are shown.</p><p>(D) Cytotoxicity assays of selected hits from the screen. A549 cells in 96-well plates were transduced with high-titer lentiviral stocks, yielding 90% transduced cells. Cells were then assayed by HTM for (from top to bottom): cell survival/proliferation, by DAPI stain and cell count; apoptosis, by Annexin V stain; DNA damage, by staining for phosphorylated H2AX; and cell membrane integrity, by Sytox stain. Tamoxifen and staurosporine were used as positive controls, untreated and untransduced cells as negative controls. Representative images from the respective assays are shown on the right.</p></caption><graphic xlink:href="figs1"></graphic></fig><fig id="figs2"><label>Figure S2</label><caption><p><italic>SERPINE1</italic> Gene Expression Profiles and the Role of Extracellular PAI-1 Protein in IAV Inhibition, Related to <xref rid="fig2" ref-type="fig">Figure 2</xref></p><p>(A–C) Extended <xref rid="fig2" ref-type="fig">Figure 2</xref>A. <italic>ISG15</italic>, <italic>IFITM3</italic>, and <italic>BST2</italic> mRNA expression was normalized to housekeeping gene <italic>RPS-11</italic>, fold increase over pre-treatment control levels is shown.</p><p>(D–F) Extended <xref rid="fig2" ref-type="fig">Figure 2</xref> A-C, showing the effect of TGF-β on <italic>SERPINE1</italic> mRNA expression (A), or on intracellular (B) and extracellular (C) PAI-1 protein levels. (G). Extended <xref rid="fig2" ref-type="fig">Figure 2</xref>D, showing the amount of extracellular PAI-1 protein levels from HAEC after induction with TGF-β. (H). A549 cells were infected with HPIV3-GFP for 48 hpi in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM. The number of infected cells was normalized to buffer (1) or IgG control (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests. (I). HAEC were infected with HPIV3-GFP in the presence of either recombinant stable human PAI-1 (rPAI-1, 1) or α-PAI-1 antibody (2), and buffer (1) or IgG (2) as controls. Progeny virus was collected from apical washes at the indicated times and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by TCID<sub>50</sub> assay. Data are represented as mean ± SEM from n = 3 replicates.</p></caption><graphic xlink:href="figs2"></graphic></fig><fig id="figs3"><label>Figure S3</label><caption><p>Effect of PAI-1 on Various Steps of the IAV Life Cycle</p><p>(A–E) A549 cells were transduced to express the indicated ISGs. Empty vector served as negative control, and the following positive controls were used: Diphyllin for IAV entry, Ribavirin for IAV replication. Data are represented as mean ± SEM from at least n = 4 independent experiments for all panels.</p><p>(A) Cells were challenged with IAV WSN/33 at MOI 1 and the number of NP-positive nuclei was determined at 6 hpi. Statistics by 1-way ANOVA and Dunn’s multiple comparison test.</p><p>(B) IAV replication efficiency was assayed by a luciferase-based IAV minigenome assay. Expression constructs for components of the IAV replication machinery (PB1, PB2, PA and NP, of A/WSN/33 origin) were co-transfected with a reporter construct mimicking the viral genome, leading to expression of firefly luciferase (Fluc) when the genome mimic is replicated. Individual t tests compared to empty control.</p><p>(C) Influenza A/Puerto Rico/8/34-NS1-GFP virus replication during infection was assayed by FACS assay, determining the percentage of infected cells at 10 hpi (GFP-positive) in the ISG-overexpressing (RFP-positive) population. Statistical significance relative to empty control was determined by t tests.</p><p>(D–F) Cells were infected with IAV WSN/33 at MOI = 0.01. At 24 hpi, vRNA was extracted from cells (D) or supernatants (E), and vRNA copy number was determined by qRT-PCR. Infectious virus titers in supernatants at 48 hpi were determined by plaque assay on MDCK cells (F).</p></caption><graphic xlink:href="figs3"></graphic></fig><fig id="figs4"><label>Figure S4</label><caption><p>Protease Targets of PAI-1, Related to <xref rid="fig4" ref-type="fig">Figure 4</xref></p><p>(A–D) Representative gel shift assays to test the formation of SDS-stable complexes of recombinant PAI-1 (rPAI-1) and furin. (A+B). 1μg of rPAI-1 was combined with increasing amounts of active recombinant furin (from abcam, A, and from NEB, B), and the mixture was separated on SDS gels followed by silver staining. Where indicated, Triplaxtinin was used at 3.3 mM to inhibit PAI-1. (C). To establish conditions for furin-rPAI-binding, 1μg rPAI was combined with 0.1 U furin (abcam) in 100 mM HEPES [pH 7.5], 1 mM CaCl<sub>2</sub>, with the addition 0.5% Triton X-100 at either 24 or 37°C for indicated times.</p><p>(D) 1μg rPAI was combined with 0.1 U furin (abcam) in 100 mM HEPES [pH 9], 1 mM CaCl<sub>2</sub>, with the addition 0.5% Triton X-100 at either 24 or 37°C for indicated times.</p></caption><graphic xlink:href="figs4"></graphic></fig><fig id="figs5"><label>Figure S5</label><caption><p>Role of <italic>Serpine1</italic> on Virus Infection in Mice, Related to <xref rid="fig6" ref-type="fig">Figure 6</xref></p><p>(A) Wild-type (B6) or <italic>Serpine1</italic><sup>−/−</sup> mice were infected intranasally with 1x10<sup>5</sup> PFU of vesicular stomatitis virus, and monitored for weight loss and survival. Data are represented as mean ± SEM from n = 10 infected B6 or <italic>Serpine1</italic><sup>−/−</sup> mice, and n = 3 respective PBS control animals. Statistical significance was determined by individual t test or log rank test. No significant differences could be detected.</p><p>(B) Wild-type (B6), <italic>Serpine</italic><sup><italic>+</italic>/−</sup> or <italic>Serpine1</italic><sup>−/−</sup> littermate mice were infected intranasally with 1000 PFU (36 LD<sub>50</sub>) of influenza A/Puerto Rico/8/34 virus, and assayed for weight loss and survival. Homogenates of infected mouse lungs were assayed for mPAI-1 protein levels by ELISA.</p><p>(C) Pathology of lung cross-sections from mouse lungs at 4 dpi, stained with hematoxylin and eosin. Bar represents 50 μm. Severity of bleeding was scored quantitatively from n = 5 mice per group.</p><p>(D) Wild-type (B6) or <italic>Serpine1</italic><sup>−/−</sup> mice were infected intranasally with 1000 PFU (36 LD<sub>50</sub>) of influenza A/Puerto Rico/8/34 virus. Homogenates of infected mouse lungs were assayed for murine <italic>RSAD2</italic> (viperin), <italic>TNF-α</italic>, <italic>IFN-β</italic>, <italic>TGF-β</italic>, <italic>MIP-2</italic> and <italic>IL-6</italic> mRNA levels by qRT-PCR. n = 5 mice per group per day. Statistical significance between groups was determined by t tests.</p><p>(E) Wild-type (B6) or <italic>Serpine1</italic><sup>−/−</sup> mice were challenged intranasally with 100μg poly(I:C). Homogenates of mouse lungs were assayed for murine <italic>RSAD2</italic> (viperin), <italic>TNF-α</italic>, <italic>IFN-β</italic>, <italic>TGF-β</italic>, <italic>MIP-2</italic> and <italic>IL-6</italic> mRNA levels by qRT-PCR. n = 5 mice per group per day. Statistical significance between groups was determined by t tests.</p></caption><graphic xlink:href="figs5"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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