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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Adaptive Evolution of MERS-CoV to Species Variation in DPP4</title><author><name sortKey="Letko, Michael" sort="Letko, Michael" uniqKey="Letko M" first="Michael" last="Letko">Michael Letko</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Miazgowicz, Kerri" sort="Miazgowicz, Kerri" uniqKey="Miazgowicz K" first="Kerri" last="Miazgowicz">Kerri Miazgowicz</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA</nlm:aff></affiliation></author><author><name sortKey="Mcminn, Rebekah" sort="Mcminn, Rebekah" uniqKey="Mcminn R" first="Rebekah" last="Mcminn">Rebekah Mcminn</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Department of Microbiology, Immunology, & Pathology, Colorado State University, Fort Collins, CO 80523, USA</nlm:aff></affiliation></author><author><name sortKey="Seifert, Stephanie N" sort="Seifert, Stephanie N" uniqKey="Seifert S" first="Stephanie N." last="Seifert">Stephanie N. Seifert</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Sola, Isabel" sort="Sola, Isabel" uniqKey="Sola I" first="Isabel" last="Sola">Isabel Sola</name><affiliation><nlm:aff id="aff5">Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain</nlm:aff></affiliation></author><author><name sortKey="Enjuanes, Luis" sort="Enjuanes, Luis" uniqKey="Enjuanes L" first="Luis" last="Enjuanes">Luis Enjuanes</name><affiliation><nlm:aff id="aff5">Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain</nlm:aff></affiliation></author><author><name sortKey="Carmody, Aaron" sort="Carmody, Aaron" uniqKey="Carmody A" first="Aaron" last="Carmody">Aaron Carmody</name><affiliation><nlm:aff id="aff2">Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Van Doremalen, Neeltje" sort="Van Doremalen, Neeltje" uniqKey="Van Doremalen N" first="Neeltje" last="Van Doremalen">Neeltje Van Doremalen</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Munster, Vincent" sort="Munster, Vincent" uniqKey="Munster V" first="Vincent" last="Munster">Vincent Munster</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">30110630</idno><idno type="pmc">7104223</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104223</idno><idno type="RBID">PMC:7104223</idno><idno type="doi">10.1016/j.celrep.2018.07.045</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000665</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000665</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Adaptive Evolution of MERS-CoV to Species Variation in DPP4</title><author><name sortKey="Letko, Michael" sort="Letko, Michael" uniqKey="Letko M" first="Michael" last="Letko">Michael Letko</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Miazgowicz, Kerri" sort="Miazgowicz, Kerri" uniqKey="Miazgowicz K" first="Kerri" last="Miazgowicz">Kerri Miazgowicz</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA</nlm:aff></affiliation></author><author><name sortKey="Mcminn, Rebekah" sort="Mcminn, Rebekah" uniqKey="Mcminn R" first="Rebekah" last="Mcminn">Rebekah Mcminn</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Department of Microbiology, Immunology, & Pathology, Colorado State University, Fort Collins, CO 80523, USA</nlm:aff></affiliation></author><author><name sortKey="Seifert, Stephanie N" sort="Seifert, Stephanie N" uniqKey="Seifert S" first="Stephanie N." last="Seifert">Stephanie N. Seifert</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Sola, Isabel" sort="Sola, Isabel" uniqKey="Sola I" first="Isabel" last="Sola">Isabel Sola</name><affiliation><nlm:aff id="aff5">Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain</nlm:aff></affiliation></author><author><name sortKey="Enjuanes, Luis" sort="Enjuanes, Luis" uniqKey="Enjuanes L" first="Luis" last="Enjuanes">Luis Enjuanes</name><affiliation><nlm:aff id="aff5">Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain</nlm:aff></affiliation></author><author><name sortKey="Carmody, Aaron" sort="Carmody, Aaron" uniqKey="Carmody A" first="Aaron" last="Carmody">Aaron Carmody</name><affiliation><nlm:aff id="aff2">Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Van Doremalen, Neeltje" sort="Van Doremalen, Neeltje" uniqKey="Van Doremalen N" first="Neeltje" last="Van Doremalen">Neeltje Van Doremalen</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author><author><name sortKey="Munster, Vincent" sort="Munster, Vincent" uniqKey="Munster V" first="Vincent" last="Munster">Vincent Munster</name><affiliation><nlm:aff id="aff1">Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Cell Reports</title><idno type="eISSN">2211-1247</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Adney, D R" uniqKey="Adney D">D.R. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell Rep</journal-id><journal-id journal-id-type="iso-abbrev">Cell Rep</journal-id><journal-title-group><journal-title>Cell Reports</journal-title></journal-title-group><issn pub-type="epub">2211-1247</issn><publisher><publisher-name>Cell Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">30110630</article-id><article-id pub-id-type="pmc">7104223</article-id><article-id pub-id-type="publisher-id">S2211-1247(18)31148-3</article-id><article-id pub-id-type="doi">10.1016/j.celrep.2018.07.045</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Adaptive Evolution of MERS-CoV to Species Variation in DPP4</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Letko</surname><given-names>Michael</given-names></name><email>michael.letko@nih.gov</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="fn1" ref-type="fn">6</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Miazgowicz</surname><given-names>Kerri</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff3" ref-type="aff">3</xref><xref rid="fn1" ref-type="fn">6</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>McMinn</surname><given-names>Rebekah</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Seifert</surname><given-names>Stephanie N.</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Sola</surname><given-names>Isabel</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Enjuanes</surname><given-names>Luis</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Carmody</surname><given-names>Aaron</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author" id="au8"><name><surname>van Doremalen</surname><given-names>Neeltje</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author" id="au9"><name><surname>Munster</surname><given-names>Vincent</given-names></name><email>vincent.munster@nih.gov</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="fn2" ref-type="fn">7</xref><xref rid="cor2" ref-type="corresp">∗∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</aff><aff id="aff2"><label>2</label>Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA</aff><aff id="aff3"><label>3</label>Department of Infectious Diseases, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA</aff><aff id="aff4"><label>4</label>Department of Microbiology, Immunology, & Pathology, Colorado State University, Fort Collins, CO 80523, USA</aff><aff id="aff5"><label>5</label>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>michael.letko@nih.gov</email></corresp><corresp id="cor2"><label>∗∗</label>Corresponding author <email>vincent.munster@nih.gov</email></corresp><fn id="fn1"><label>6</label><p id="ntpara0010">These authors contributed equally</p></fn><fn id="fn2"><label>7</label><p id="ntpara0015">Lead Contact</p></fn></author-notes><pub-date pub-type="pmc-release"><day>14</day><month>8</month><year>2018</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>14</day><month>8</month><year>2018</year></pub-date><pub-date pub-type="epub"><day>14</day><month>8</month><year>2018</year></pub-date><volume>24</volume><issue>7</issue><fpage>1730</fpage><lpage>1737</lpage><history><date date-type="received"><day>3</day><month>4</month><year>2018</year></date><date date-type="rev-recd"><day>13</day><month>6</month><year>2018</year></date><date date-type="accepted"><day>12</day><month>7</month><year>2018</year></date></history><permissions><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract id="abs0010"><title>Summary</title><p>Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4.</p></abstract><abstract abstract-type="graphical" id="abs0015"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1_lrg"></graphic></fig></abstract><abstract abstract-type="author-highlights" id="abs0020"><title>Highlights</title><p><list list-type="simple" id="ulist0010"><list-item id="u0010"><label>•</label><p id="p0010">MERS-CoV infected cells expressing DPP4 from 16 bat species</p></list-item><list-item id="u0015"><label>•</label><p id="p0015">MERS-CoV spike rapidly adapted to species variation in DPP4</p></list-item><list-item id="u0020"><label>•</label><p id="p0020">Viral adaptations modified the surface charge of viral spike</p></list-item><list-item id="u0025"><label>•</label><p id="p0025">Different routes of spike adaptation enhanced entry with the same DPP4 variant</p></list-item></list></p></abstract><abstract abstract-type="teaser" id="abs0025"><p>MERS-CoV is a zoonotic pathogen capable of infecting numerous species. However, our understanding of how this virus adapts to new species remains unclear. Letko et al. experimentally observe several different routes in the stepwise, adaptive evolution of MERS-CoV to a unique host-species variant of the viral receptor.</p></abstract><kwd-group id="kwrds0010"><title>Keywords</title><kwd>MERS</kwd><kwd>Coronavirus</kwd><kwd>Spike</kwd><kwd>DPP4</kwd><kwd>Dipeptidyl peptidase IV</kwd><kwd>Evolution</kwd><kwd>Zoonosis</kwd><kwd>Species barrier</kwd><kwd>Adaptation</kwd><kwd>Bat</kwd><kwd>Desmodus rotundus</kwd></kwd-group></article-meta><notes><p id="misc0010">Published: August 14, 2018</p></notes></front><body><sec id="sec1"><title>Introduction</title><p id="p0030">Since its discovery in 2012, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has infected over 2,000 people and has a mortality rate of approximately 35% (<xref rid="bib33" ref-type="bibr">World Health Organization, 2018</xref>WHO; <xref rid="bib37" ref-type="bibr">Zaki et al., 2012</xref>). The ancestral host species of MERS-CoV remains elusive; however, mounting evidence suggests that the virus most likely originated in bats and passed to humans through multiple zoonotic spill-over events from dromedary camels (<xref rid="bib4" ref-type="bibr">Azhar et al., 2014</xref>, <xref rid="bib15" ref-type="bibr">Haagmans et al., 2014</xref>, <xref rid="bib16" ref-type="bibr">Ithete et al., 2013</xref>, <xref rid="bib34" ref-type="bibr">Woo et al., 2012</xref>). While species capable of supporting MERS-CoV infection have been studied, the genetic mechanisms underlying cross-species adaptation remain poorly understood.</p><p id="p0035">A primary determinant of viral species-tropism is at the level of host cell entry, which is mediated by MERS-CoV spike protein binding host dipeptidyl peptidase 4 (DPP4) (<xref rid="bib22" ref-type="bibr">Ohnuma et al., 2013</xref>, <xref rid="bib25" ref-type="bibr">Raj et al., 2013</xref>). Structural studies have shown that this interaction relies on multiple contact points (<xref rid="bib19" ref-type="bibr">Lu et al., 2013</xref>, <xref rid="bib28" ref-type="bibr">Song et al., 2014</xref>, <xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>). Our group has previously demonstrated that hamster DPP4 contains variation in these contact points, which prevents MERS-CoV replication (<xref rid="bib30" ref-type="bibr">van Doremalen et al., 2014</xref>, <xref rid="bib31" ref-type="bibr">van Doremalen et al., 2016</xref>). Other genetic variation responsible for the glycosylation of DPP4 in mice, hamsters, ferrets, and guinea pigs forms an additional block to the interaction with MERS-CoV spike (<xref rid="bib9" ref-type="bibr">Cockrell et al., 2014</xref>, <xref rid="bib24" ref-type="bibr">Peck et al., 2017</xref>). In contrast to small rodents and ferrets, MERS-CoV is capable of utilizing DPP4 from different bat species, and <italic>Artibeus jamaicensis</italic> bats have been shown to support experimental infection (<xref rid="bib8" ref-type="bibr">Caì et al., 2014</xref>, <xref rid="bib21" ref-type="bibr">Munster et al., 2016</xref>).</p><p id="p0040">Here, we investigated the host breadth and adaptability of MERS-CoV. We demonstrate that MERS-CoV can use DPP4 from a diverse range of bat species and can rapidly adapt to variation in DPP4.</p></sec><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>Identification of a Semi-Permissive DPP4 Variant</title><p id="p0045">We selected 16 bat species representing a broad taxonomic and geographic distribution to screen for MERS-CoV entry (<xref rid="fig1" ref-type="fig">Figures 1</xref>A, <xref rid="mmc1" ref-type="supplementary-material">S1</xref>, and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>). Plasmids encoding the bat DPP4s were transfected into Baby Hamster Kidney fibroblasts (BHK) cells, which are non-permissive to MERS-CoV infection (<xref rid="bib30" ref-type="bibr">van Doremalen et al., 2014</xref>). All DPP4s supported viral replication to varying degrees; however, <italic>Desmodus rotundus</italic> DPP4 (<italic>dr</italic>DPP4) was the least permissive to viral replication (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). Flow cytometry showed that DPP4 surface expression varied between species but did not correlate with infectivity (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>A). While this may result from variation in antibody recognition, to date, no commercially available DPP4 antibody recognizes an epitope that is conserved across all species.<fig id="fig1"><label>Figure 1</label><caption><p><italic>Desmodus Rotundus</italic> DPP4 Is Semi-Permissive for MERS-CoV Replication</p><p>(A) Phylogenetic tree of the DPP4 amino acid sequences used in this study. See also <xref rid="mmc1" ref-type="supplementary-material">Figures S1</xref> and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>.</p><p>(B) BHK cells were transfected with equivalent amounts of bat-DPP4 expression plasmids. Cells were infected 24 hr later with MERS-CoV at MOI = 1. Viral titer of 48-hr supernatants was determined by titration on Vero cells. Each dot on the graph represents one replicate and horizontal black lines represent the mean of three replicates. See also <xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>A.</p><p>(C) Comparison of MERS-spike binding residues between human and <italic>Desmodus rotundus</italic> DPP4. See also <xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>B.</p><p>(D) Structure of MERS-CoV spike protein bound to human DPP4 (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4L72" id="intref0035">4L72</ext-link>) with DPP4 residues 295 and 317 indicated in yellow and red, respectively.</p><p>(E) Human and <italic>dr</italic>DPP4-295 and 317 mutants were transfected in BHK cells. Cells were infected 24 hr later with MERS-CoV at MOI = 1. Viral titer of 48-hr supernatants was determined by titration on Vero cells. Each dot on the graph represents one replicate, and horizontal black lines represent the mean of three replicates.</p></caption><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0050">Relative to human DPP4, the bat DPP4s contained variation throughout the whole spike-binding region (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>B). However, within the residues, which specifically bind MERS-CoV spike, <italic>dr</italic>DPP4 only differed from human DPP4 in two amino acids at positions 295 and 317 (<xref rid="fig1" ref-type="fig">Figures 1</xref>C, 1D, and <xref rid="mmc1" ref-type="supplementary-material">S3</xref>B) (<xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>). As <italic>dr</italic>DPP4 was also unique at position 317 (R317Q), we chose to focus on <italic>dr</italic>DPP4. Introducing <italic>dr</italic>DPP4 mutations into human DPP4 reduced viral replication, while mutating <italic>dr</italic>DPP4 to match the amino acids in human DPP4 increased its ability to support MERS-CoV replication (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). These data show that residues 295 and 317 largely determine resistance of <italic>dr</italic>DPP4 to MERS-CoV replication.</p></sec><sec id="sec2.2"><title>Viral Adaptation of MERS-CoV to <italic>Desmodus Rotundus</italic> DPP4</title><p id="p0055">As only two <italic>dr</italic>DPP4 residues influenced MERS-CoV replication, we hypothesized that viral spike may be able to adapt to this variation. To test this, we generated BHK cell lines that stably expressed human DPP4, <italic>dr</italic>DPP4, or blue fluorescent protein (BFP; a negative control) from a lentiviral expression cassette that also included a fluorescent reporter, mCherry, as well as a puromycin selection gene (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). These genes were separated by 2A sequences, which allowed for all three to express under the human Ef1α promoter (<xref rid="bib17" ref-type="bibr">Letko et al., 2015</xref>). Flow cytometry for mCherry and DPP4 confirmed similar transduction efficiency and transgene expression levels, respectively, between human and <italic>dr</italic>DPP4 cell lines (<xref rid="fig2" ref-type="fig">Figure 2</xref>B). Cells expressing <italic>dr</italic>DPP4 were less susceptible than human DPP4 to MERS-CoV infection, similar to our transfection experiments (<xref rid="fig2" ref-type="fig">Figure 2</xref>C).<fig id="fig2"><label>Figure 2</label><caption><p>Forced Adaptation of MERS-CoV on DPP4 Stable Cell Lines</p><p>(A) An overview of the lentiviral expression cassette.</p><p>(B) Flow cytometry of transduced cells.</p><p>(C) Transduced cells were infected with MERS-CoV at MOI = 0.001. Viral titer was determined by titration on Vero cells. Error bars represent the SDs of three replicates.</p><p>(D) MERS-CoV was passaged on DPP4-tansduced cells. Cytopathic effects were observed by passage 8.</p><p>(E) Sequencing chromatograms of MERS-CoV spike from different passages on the <italic>dr</italic>DPP4 cells. Arrows indicate emerging mutations (overlapping peaks).</p><p>(F) Schematic of single and double mutation emergence in MERS-CoV spike over different passages. See also <xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>A.</p><p>(G) Location of mutations within MERS-CoV spike.</p><p>(H) Location of mutations in the MERS-CoV spike co-structure with human DPP4 (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4L72" id="intref0040">4L72</ext-link>). MERS-CoV spike is colored blue and residues 465 and 510 are shown in pink and orange, respectively. DPP4 is colored gray and residues 295 and 317 are shown in yellow and red, respectively.</p></caption><graphic xlink:href="gr2_lrg"></graphic></fig></p><p id="p0060">Wild-type (WT) MERS-CoV was serially passaged, in triplicate, on these DPP4 and control cell lines. While human DPP4 cells showed cytopathic effects (CPE) from the first passage, <italic>dr</italic>DPP4 cells only showed CPE after eight passages, indicative of viral adaptation (<xref rid="fig2" ref-type="fig">Figure 2</xref>D). Sanger sequencing of the MERS-CoV spike receptor binding domain (RBD) revealed the emergence of several mutations with the <italic>dr</italic>DPP4 cell cultures (<xref rid="fig2" ref-type="fig">Figure 2</xref>E). By the fifth passage, spike S465F emerged in two replicates, and spike D510H emerged in one replicate (<xref rid="fig2" ref-type="fig">Figure 2</xref>F). By passage 8, one S465F mutant acquired a secondary D510G mutation, and on passage 12, the D510H mutant acquired a secondary S465F mutation (<xref rid="fig2" ref-type="fig">Figure 2</xref>F). We sequenced the spike RBD from 14 passages, as well as full-length spike from passages 3, 6, and 9, but did not observe additional mutations. No spike mutations were observed with the human DPP4 or control cell lines, indicating that these mutations were specific for <italic>dr</italic>DPP4 and not the BHK cells themselves. Spike residues 465 and 510 are located within the RBD and interface with DPP4 (<xref rid="fig2" ref-type="fig">Figures 2</xref>G and 2H). Notably, spike residue 510 clusters with DPP4 residues 295 and 317 and directly interacts with DPP4 residue 317 (<xref rid="fig2" ref-type="fig">Figure 2</xref>H) (<xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>). MERS spike 465S and 510D are found in the majority of published spike sequences (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>A).</p></sec><sec id="sec2.3"><title>Characterization of MERS-CoV Spike Mutations</title><p id="p0065">Human and <italic>dr</italic>DPP4 are 83.8% similar at the amino acid level and both function as the receptor for MERS-CoV, suggesting the two proteins share a common structure. Therefore, we used structural data for human DPP4 to predict the structure of <italic>dr</italic>DPP4. Electrostatic potential analysis showed a positive surface charge of human DPP4 at residues 295 and 317 and a negative charge at these positions in <italic>dr</italic>DPP4 (<xref rid="fig3" ref-type="fig">Figure 3</xref>A). Complementary to human DPP4, the WT MERS-CoV spike RBD is negatively charged (<xref rid="fig3" ref-type="fig">Figure 3</xref>B). This analysis revealed that the spike adaptations altered the surface charge of spike from negative to positive, complementing the negative charge of <italic>dr</italic>DPP4 (<xref rid="fig3" ref-type="fig">Figure 3</xref>C).<fig id="fig3"><label>Figure 3</label><caption><p>Structural Analysis and Functional Testing of Spike Mutations</p><p>(A) Surface charge of human DPP4 (amino acids 39–766; PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4L72" id="intref0045">4L72</ext-link>) and predicted <italic>dr</italic>DPP4 structure (amino acids 37–765). Residues 295 and 317 are shown. Blue indicates positive charge, red indicates negative charge.</p><p>(B) Surface charge of the MERS-CoV spike (amino acids 382–585; PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4L72" id="intref0050">4L72</ext-link>).</p><p>(C) Surface charge of MERS-CoV spike mutations.</p><p>(D) DPP4 cells were infected with mutant viruses at MOI = 0.001 and supernatants were collected at the indicated time points. Viral titer was determined qRT-PCR. Error bars represent the SDs of three replicates. See also <xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>B.</p><p>(E) DPP4 transduced cells were infected with spike-pseudotyped VSV-particles and luciferase was measured 24 hr later. Error bars represent the SDs of three replicates.</p></caption><graphic xlink:href="gr3_lrg"></graphic></fig></p><p id="p0070">Because we only sequenced MERS-CoV spike, it remained possible that other mutations arose in the viral genome that increased viral replication in the <italic>dr</italic>DPP4 cells. Therefore, we generated spike point mutant viruses, which failed to grow on control transduced cells but grew to similar levels as WT virus and induced CPE in the human DPP4 cells (<xref rid="fig3" ref-type="fig">Figures 3</xref>D and S4B). In contrast, WT virus replicated to lower titers than the spike mutants and failed to induce CPE in <italic>dr</italic>DPP4 cells (<xref rid="fig3" ref-type="fig">Figures 3</xref>D and <xref rid="mmc1" ref-type="supplementary-material">S4</xref>B). Next, we performed an entry assay with spike-pseudotyped vesicular stomatitis virus (VSV) particles expressing luciferase. While all spike mutations slightly increased entry over WT spike on human DPP4 cells, there was a striking increase in entry with <italic>dr</italic>DPP4 cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>E). Collectively, these findings demonstrate that adaptation in MERS-CoV spike enhanced viral replication on <italic>dr</italic>DPP4 cells through increased viral entry.</p></sec><sec id="sec2.4"><title>Spike Adaptation Specificity</title><p id="p0075">We transfected cells with a subset of the bat DPP4 panel and then infected with spike mutant viruses, all of which replicated better than WT virus on cells expressing <italic>dr</italic>DPP4 (<xref rid="fig4" ref-type="fig">Figure 4</xref>). While some mutants performed better than WT virus on cells expressing other bat DPP4s, there was not a consistent top-performing mutant. Notably, spike-510H only replicated better than WT virus with <italic>dr</italic>DPP4 and not the other bat DPP4s, suggesting that this mutation is specific for <italic>dr</italic>DPP4.<fig id="fig4"><label>Figure 4</label><caption><p>Spike Adaptation Specificity</p><p>BHKs were transfected with indicated DPP4s and infected with mutant viruses at MOI = 0.005. Viral titer of supernatants taken at 72 hr was determined by qRT-PCR. Error bars represent SDs of two replicates.</p></caption><graphic xlink:href="gr4_lrg"></graphic></fig></p></sec></sec><sec id="sec3"><title>Discussion</title><p id="p0080">While numerous studies have shown that MERS-CoV can infect several different species, our understanding of the genetic mechanisms underlying cross species spillover remains unclear (<xref rid="bib1" ref-type="bibr">Adney et al., 2014</xref>, <xref rid="bib2" ref-type="bibr">Agrawal et al., 2015</xref>, <xref rid="bib10" ref-type="bibr">Cockrell et al., 2016</xref>, <xref rid="bib12" ref-type="bibr">de Wit et al., 2013</xref>, <xref rid="bib14" ref-type="bibr">Falzarano et al., 2014</xref>, <xref rid="bib21" ref-type="bibr">Munster et al., 2016</xref>). Here, we investigated species promiscuity and adaptability of MERS-CoV.</p><p id="p0085">Previous studies have shown that MERS-CoV entry is less efficient with DPP4 from pipistrelle bats compared to human DPP4 (<xref rid="bib7" ref-type="bibr">Barlan et al., 2014</xref>, <xref rid="bib25" ref-type="bibr">Raj et al., 2013</xref>, <xref rid="bib36" ref-type="bibr">Yang et al., 2014</xref>). This difference in receptor efficiency was used to suggest that MERS-CoV spike adapted away from using bat DPP4 as it adapted to human DPP4. While our data confirm this replication phenotype with <italic>Pipistrellus</italic> DPP4, we showed that DPP4 from several other bat species supported MERS-CoV replication comparable to or better than human DPP4 (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). Thus, without more evidence as to the true ancestral host of MERS-CoV, conclusions regarding spike adaptation away from bats should be more reserved. This finding also highlights the importance of screening a broad selection of species when assessing host breadth.</p><p id="p0090"><italic>A. planirostris</italic> and <italic>A. jamaicensis</italic> DPP4s are identical to human DPP4 at the 14 contact points with spike but differed in their ability to support MERS-CoV replication (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). Notably, these two bat DPP4 sequences varied from human DPP4 and from each other at other positions in the receptor binding domain (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>B), suggesting that additional DPP4 residues can influence the interaction with spike.</p><p id="p0095">Forced adaptation of MERS-CoV spike has been a challenge in the field as DPP4 from most non-permissive species contains glycosylation that completely abrogates the interaction and blocks infection (<xref rid="bib9" ref-type="bibr">Cockrell et al., 2014</xref>, <xref rid="bib23" ref-type="bibr">Peck et al., 2015</xref>, <xref rid="bib24" ref-type="bibr">Peck et al., 2017</xref>). Without viral replication, it has not been possible to experimentally demonstrate evolution of the MERS-CoV spike. We found that <italic>dr</italic>DPP4 was only semi-permissive to viral infection and, therefore, served as a tool to test MERS-CoV spike adaptation <italic>in vitro</italic> (<xref rid="fig1" ref-type="fig">Figures 1</xref> and <xref rid="fig2" ref-type="fig">2</xref>). Within three passages on cells expressing <italic>dr</italic>DPP4, MERS-CoV accumulated mutations in the receptor-binding domain of spike that enhanced viral entry and replication specifically with <italic>dr</italic>DPP4 and not the other bat DPP4s (<xref rid="fig2" ref-type="fig">Figures 2</xref>, <xref rid="fig3" ref-type="fig">3</xref>, and <xref rid="fig4" ref-type="fig">4</xref>). The prevalence of these adaptation mutations is low in published sequences, further suggesting that they are specific for <italic>dr</italic>DPP4 (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>A). Taken together, MERS-CoV spike is likely adaptable to any DPP4 variation that does not completely ablate the interaction.</p><p id="p0100">At least 14 contact points have been identified in crystal structures of human DPP4 and MERS-CoV spike (<xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>). <italic>dr</italic>DPP4 contains variation within and around these known points (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>B). Therefore, the spike-465F mutation, which is just outside of the interface, may lead to broader structural adjustments that favor interaction with <italic>dr</italic>DPP4 (<xref rid="fig2" ref-type="fig">Figure 2</xref>H). Surprisingly, the adaptation mutations had very little effect on the interaction with human DPP4, suggesting that other contact points could compensate for this variation (<xref rid="fig3" ref-type="fig">Figure 3</xref>). For example, spike residue 510 has been shown to contact DPP4 residue 322 in addition to 317, so it may be possible that this interaction is retained with human DPP4 (<xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>). Structural studies are needed to fully define how these spike mutations influence the interface with DPP4.</p><p id="p0105">Our viral adaptation experiments revealed mutations in MERS-CoV spike, which altered the surface charge to complement the opposite charge of <italic>dr</italic>DPP4 (<xref rid="fig3" ref-type="fig">Figure 3</xref>C). Interestingly, these mutations share a striking similarity to species adaptations identified in SARS-CoV spike (as reviewed in <xref rid="bib18" ref-type="bibr">Li, 2013</xref>). The ability to rapidly adjust surface charge may be a general property of MERS-CoV spike to facilitate cross-species adaptation. However, animal studies are needed to see if the mutations we described here affect other viral phenotypes, including host immune evasion.</p><p id="p0110">Previous work has assessed coronavirus spike adaptation to orthologous host receptors (<xref rid="bib6" ref-type="bibr">Baric et al., 1999</xref>, <xref rid="bib20" ref-type="bibr">McRoy and Baric, 2008</xref>, <xref rid="bib26" ref-type="bibr">Roberts et al., 2007</xref>, <xref rid="bib27" ref-type="bibr">Sheahan et al., 2008</xref>, <xref rid="bib35" ref-type="bibr">Wu et al., 2012</xref>). These studies characterized viruses after passage in animals, long-term cell culture, or performed biochemical analysis on spike variations from different isolates. As these experiments only focused on the viral endpoint, they failed to capture the continuous evolution through viral passages. Thus, the exact speed and order in which mutations arise in response to receptors from non-cognate species remain unclear in these studies. The work we present here demonstrates that MERS-CoV rapidly adapts to DPP4 variation and can do so utilizing different mutation paths. While <italic>D. rotundus</italic> is not known to be a host species for MERS-CoV, the use of its semi-permissive DPP4 allowed us to observe, with high temporal resolution, species adaptation of MERS-CoV. All together, these findings shed light on the evolutionary mechanisms underlying MERS-CoV cross-species transmission.</p></sec><sec id="sec4"><title>Experimental Procedures</title><sec id="sec4.1"><title>Biosafety Statement</title><p id="p0115">All work with infectious MERS-CoV was approved by the Rocky Mountain Laboratories Institutional Biosafety Committee and performed under biosafety level 3 conditions.</p></sec><sec id="sec4.2"><title>Sequencing of Bat DPP4</title><p id="p0120">cDNA from <italic>E. buettikoferi</italic> and <italic>R. aegyptiacus</italic> were derived from the EpoNi/22.1 and RE06 cell lines, respectively. cDNA was produced from <italic>A. planirostris</italic>, <italic>C. perspicillata</italic>, and <italic>S. bilineata</italic> primary lung tissue and <italic>H. gigas</italic> heart tissue, provided by Dr. Tony Schountz. Dr. Lin-Fa Wang provided cDNA extracted from <italic>R. ferrumequinum</italic> primary kidney tissue. DPP4 was amplified using the iProof High-fidelity PCR kit (Bio-Rad), following the manufacturer’s instructions and Sanger sequencing.</p></sec><sec id="sec4.3"><title>Plasmids</title><p id="p0125">DPP4 coding sequences from human (NM001935.3), <italic>A. jamaicensis</italic> (KF574262), <italic>A. planirostris</italic> (MH299895), <italic>C. perspicillata</italic> (MH299896), <italic>D. rotundus</italic> (GABZ01004546.1), <italic>E. buettikoferi</italic> (MH299897), <italic>E. fuscus</italic> (XM008138769), <italic>H. gigas</italic> (MH299898), <italic>M. brandtii</italic> (XM005859372.1), <italic>M.davidii</italic> (XM006766490.1), <italic>M. lucifugus</italic> (XM006083213.1), <italic>P. pipistrellus</italic> (KC249974.1), <italic>P. alecto</italic> (XM006921123.1), <italic>P. vampyrus</italic> (XM011358549), <italic>R. ferrumequinum</italic> (MH299899), <italic>R. aegyptiacus</italic> (MH299900), and <italic>S. bilineata</italic> (MH299901) were synthesized into pcDNA3.1 (Thermo-Fisher). DPP4 was cloned into the lentiviral expression vector provided by Dr. Viviana Simon. MERS-CoV spike plasmid was provided by Dr. Fang Li. Mutations were introduced into DPP4, pBAC-MERS/EMC12, or the MERS-spike plasmid by overlap PCR.</p></sec><sec id="sec4.4"><title>Viruses</title><p id="p0130">MERS-CoV/EMC12 was propagated as previously described (<xref rid="bib31" ref-type="bibr">van Doremalen et al., 2016</xref>). The WT virus stock was deep sequenced on the Illumina platform to confirm the absence of mutations described in this study. Mutant viruses were rescued as previously described (<xref rid="bib3" ref-type="bibr">Almazán et al., 2013</xref>). Titers of all viruses used in this study were determined by endpoint titration in Vero cells as previously described (<xref rid="bib30" ref-type="bibr">van Doremalen et al., 2014</xref>).</p></sec><sec id="sec4.5"><title>Transfections</title><p id="p0135">Cells were transfected, in 6-well format, with 3 μg of DNA using Lipofectamine 2000 (Life Technologies), following the manufacturer’s instructions.</p></sec><sec id="sec4.6"><title>Lentiviral Vectors, Cell Transduction, and Flow Cytometry</title><p id="p0140">Lentiviral particles were produced in 293T cells. Transduced BHKs were placed under 1 μg/mL puromycin selection, as described previously (<xref rid="bib17" ref-type="bibr">Letko et al., 2015</xref>). Flow cytometry was used to determine mCherry transduction efficiency and DPP4 expression (AF1180, R&D Systems).</p></sec><sec id="sec4.7"><title>Forced Adaptation of MERS-CoV EMC 2012 on Transduced Cells</title><p id="p0145">Human DPP4, <italic>dr</italic>DPP4, and BFP cells were infected with MERS-CoV/EMC12 at an MOI of 0.01 in triplicate. Every 72 hr, 250 μL of supernatant from each replicate was transferred to fresh cell cultures. For each passage, RNA was extracted from supernatant using the QIAamp Viral RNA kit (QIAGEN) and converted to cDNA using SuperScript III (Invitrogen). The spike RBD was amplified and Sanger sequenced. Full-length spike was sequenced for passages 3, 6, and 9.</p></sec><sec id="sec4.8"><title>Structural Modeling and Electrostatic Potential Analysis</title><p id="p0150">The co-structure of MERS-CoV spike and human DPP4 (PDB ID: <ext-link ext-link-type="uri" xlink:href="pdb:4L72" id="intref0010">4L72</ext-link> [<xref rid="bib32" ref-type="bibr">Wang et al., 2013</xref>]) was modeled in Pymol and used to predict the structure for <italic>dr</italic>DPP4 (<ext-link ext-link-type="uri" xlink:href="https://swissmodel.expasy.org" id="intref0015">https://swissmodel.expasy.org</ext-link>). Poisson-Boltzmann analysis was performed with the PDB2PQR server (<xref rid="bib13" ref-type="bibr">Dolinsky et al., 2004</xref>) and adaptive Poisson-Blotzmann Solver tool extension in Pymol (<xref rid="bib5" ref-type="bibr">Baker et al., 2001</xref>).</p></sec><sec id="sec4.9"><title>MERS-CoV Replication Kinetics</title><p id="p0155">Viral RNA was extracted from culture supernatants with the QIAamp viral RNA kit (QIAGEN). MERS-CoV titer of cell culture supernatants was determined by endpoint titration on Vero cells (<xref rid="bib31" ref-type="bibr">van Doremalen et al., 2016</xref>) or qRT-PCR as previously described (<xref rid="bib11" ref-type="bibr">Corman et al., 2012</xref>).</p></sec><sec id="sec4.10"><title>Pseudotype Entry Assay</title><p id="p0160">Pseudotyped VSV particles were produced as previously described and titered on Vero cells (<xref rid="bib29" ref-type="bibr">Takada et al., 1997</xref>). BHK-DPP4 cells were infected at an MOI = 1 with pseudotyped particles as previously described (<xref rid="bib36" ref-type="bibr">Yang et al., 2014</xref>). 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Med.</source><volume>367</volume><year>2012</year><fpage>1814</fpage><lpage>1820</lpage><pub-id pub-id-type="pmid">23075143</pub-id></element-citation></ref></ref-list><sec id="app1"><title>Data and Software Availability</title><p id="p0185">Accession numbers for the novel DPP4 sequences reported in this paper are GenBank: MH299895-MH299901. DPP4 sequence alignment from this study is available via Mendeley Data: <ext-link ext-link-type="uri" xlink:href="https://data.mendeley.com/datasets/232nv29t87/1" id="intref0020">https://data.mendeley.com/datasets/232nv29t87/1</ext-link>.</p></sec><sec id="app3" sec-type="supplementary-material"><title>Supplemental Information</title><p id="p0195"><supplementary-material content-type="local-data" id="mmc1"><caption><title>Document S1. Figures S1–S4</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc2"><caption><title>Document S2. Article plus Supplemental Information</title></caption><media xlink:href="mmc2.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We would like to thank Dr. Viviana Simon for providing the lentiviral system, Dr. Fang Li for providing the MERS-CoV spike plasmid, Dr. Tony Schountz for providing bat tissues, Dr. Lin-Fa Wang for providing <italic>R. ferrumequinum</italic> cDNA, and Drs. Bart Haagmans and Ron Fouchier for providing MERS-CoV/EMC12. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and a grant from the <funding-source id="gs1">U.S. National Institutes of Health</funding-source> awarded to L.E. (2P01AI060699).</p><sec id="sec5"><title>Author Contributions</title><p id="p0175">M.L., R.M., N.v.D., and V.M. designed experiments. M.L., R.M., K.M., S.N.S., A.C., N.v.D., L.E., and I.S. performed experiments. M.L. wrote the paper. All authors contributed to the study and provided comments on the manuscript.</p></sec><sec sec-type="COI-statement" id="sec6"><title>Declaration of Interests</title><p id="p0180">The authors declare no competing interests.</p></sec></ack><fn-group><fn id="app2" fn-type="supplementary-material"><p id="p0190">Supplemental Information includes four figures and can be found with this article online at <ext-link ext-link-type="doi" xlink:href="10.1016/j.celrep.2018.07.045" id="intref0025">https://doi.org/10.1016/j.celrep.2018.07.045</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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