Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection
Identifieur interne : 001249 ( Pmc/Corpus ); précédent : 001248; suivant : 001250Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection
Auteurs : Enya Qing ; Michael Hantak ; Stanley Perlman ; Tom GallagherSource :
- mBio [ 2150-7511 ] ; 2020.
Abstract
CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. These findings raise the possibility that the lectin-like properties of many CoVs contribute to facile zoonotic transmission and intercellular spread within infected organisms.
Url:
DOI: 10.1128/mBio.02764-19
PubMed: 32047128
PubMed Central: 7018658
Links to Exploration step
PMC:7018658Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection</title><author><name sortKey="Qing, Enya" sort="Qing, Enya" uniqKey="Qing E" first="Enya" last="Qing">Enya Qing</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hantak, Michael" sort="Hantak, Michael" uniqKey="Hantak M" first="Michael" last="Hantak">Michael Hantak</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Perlman, Stanley" sort="Perlman, Stanley" uniqKey="Perlman S" first="Stanley" last="Perlman">Stanley Perlman</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Gallagher, Tom" sort="Gallagher, Tom" uniqKey="Gallagher T" first="Tom" last="Gallagher">Tom Gallagher</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32047128</idno><idno type="pmc">7018658</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018658</idno><idno type="RBID">PMC:7018658</idno><idno type="doi">10.1128/mBio.02764-19</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">001249</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001249</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection</title><author><name sortKey="Qing, Enya" sort="Qing, Enya" uniqKey="Qing E" first="Enya" last="Qing">Enya Qing</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hantak, Michael" sort="Hantak, Michael" uniqKey="Hantak M" first="Michael" last="Hantak">Michael Hantak</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Perlman, Stanley" sort="Perlman, Stanley" uniqKey="Perlman S" first="Stanley" last="Perlman">Stanley Perlman</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA</addr-line></nlm:aff></affiliation></author><author><name sortKey="Gallagher, Tom" sort="Gallagher, Tom" uniqKey="Gallagher T" first="Tom" last="Gallagher">Tom Gallagher</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">mBio</title><idno type="eISSN">2150-7511</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">mBio</journal-id><journal-id journal-id-type="iso-abbrev">mBio</journal-id><journal-id journal-id-type="hwp">mbio</journal-id><journal-id journal-id-type="pmc">mbio</journal-id><journal-id journal-id-type="publisher-id">mBio</journal-id><journal-title-group><journal-title>mBio</journal-title></journal-title-group><issn pub-type="epub">2150-7511</issn><publisher><publisher-name>American Society for Microbiology</publisher-name><publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32047128</article-id><article-id pub-id-type="pmc">7018658</article-id><article-id pub-id-type="publisher-id">mBio02764-19</article-id><article-id pub-id-type="doi">10.1128/mBio.02764-19</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="overline"><subject>Host-Microbe Biology</subject></subj-group></article-categories><title-group><article-title>Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection</article-title><alt-title alt-title-type="running-head">Sialic Acids Facilitate Coronavirus Dissemination</alt-title><alt-title alt-title-type="short-authors">Qing et al.</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Qing</surname><given-names>Enya</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hantak</surname><given-names>Michael</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref><xref ref-type="author-notes" rid="fn1">*</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">https://orcid.org/0000-0003-4213-2354</contrib-id><name><surname>Perlman</surname><given-names>Stanley</given-names></name><xref ref-type="aff" rid="aff2"><sup>b</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Gallagher</surname><given-names>Tom</given-names></name><xref ref-type="aff" rid="aff1"><sup>a</sup></xref></contrib><aff id="aff1"><label>a</label><addr-line>Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA</addr-line></aff><aff id="aff2"><label>b</label><addr-line>Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA</addr-line></aff></contrib-group><contrib-group><contrib contrib-type="editor"><name><surname>Denison</surname><given-names>Mark R.</given-names></name><role>Editor</role><aff>Vanderbilt University Medical Center</aff></contrib></contrib-group><author-notes><corresp id="cor1">Address correspondence to Tom Gallagher, <email>tgallag@luc.edu</email>.</corresp><fn id="fn1" fn-type="present-address"><label>*</label><p>Present address: Michael Hantak, Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, USA.</p></fn></author-notes><pub-date pub-type="epub"><day>11</day><month>2</month><year>2020</year></pub-date><pub-date pub-type="collection"><season>Jan-Feb</season><year>2020</year></pub-date><volume>11</volume><issue>1</issue><elocation-id>e02764-19</elocation-id><history><date date-type="received"><day>15</day><month>10</month><year>2019</year></date><date date-type="accepted"><day>21</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="mBio.02764-19.pdf"></self-uri><abstract abstract-type="precis"><p>CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. These findings raise the possibility that the lectin-like properties of many CoVs contribute to facile zoonotic transmission and intercellular spread within infected organisms.</p></abstract><abstract><title>ABSTRACT</title><p>Coronaviruses (CoVs) are common human and animal pathogens that can transmit zoonotically and cause severe respiratory disease syndromes. CoV infection requires spike proteins, which bind viruses to host cell receptors and catalyze virus-cell membrane fusion. Several CoV strains have spike proteins with two receptor-binding domains, an S1A that engages host sialic acids and an S1B that recognizes host transmembrane proteins. As this bivalent binding may enable broad zoonotic CoV infection, we aimed to identify roles for each receptor in distinct infection stages. Focusing on two betacoronaviruses, murine JHM-CoV and human Middle East respiratory syndrome coronavirus (MERS-CoV), we found that virus particle binding to cells was mediated by sialic acids; however, the transmembrane protein receptors were required for a subsequent virus infection. These results favored a two-step process in which viruses first adhere to sialic acids and then require subsequent engagement with protein receptors during infectious cell entry. However, sialic acids sufficiently facilitated the later stages of virus spread through cell-cell membrane fusion, without requiring protein receptors. This virus spread in the absence of the prototype protein receptors was increased by adaptive S1A mutations. Overall, these findings reveal roles for sialic acids in virus-cell binding, viral spike protein-directed cell-cell fusion, and resultant spread of CoV infections.</p></abstract><kwd-group><title>KEYWORDS</title><kwd>coronavirus</kwd><kwd>virus entry</kwd><kwd>virus receptors</kwd><kwd>virus glycoproteins</kwd><kwd>sialic acids</kwd><kwd>membrane fusion</kwd></kwd-group><funding-group><award-group id="award1"><funding-source><institution-wrap><institution>HHS | National Institutes of Health (NIH)</institution><institution-id>https://doi.org/10.13039/100000002</institution-id></institution-wrap></funding-source><award-id>P01 AI060699</award-id><principal-award-recipient><name><surname>Gallagher</surname><given-names>Tom</given-names></name></principal-award-recipient><principal-award-recipient><name><surname>Perlman</surname><given-names>Stanley</given-names></name></principal-award-recipient><principal-award-recipient><name><surname>Qing</surname><given-names>Enya</given-names></name></principal-award-recipient><principal-award-recipient><name><surname>Hantak</surname><given-names>Michael</given-names></name></principal-award-recipient></award-group></funding-group><counts><count count="5" count-type="supplementary-material"></count><fig-count count="7"></fig-count><table-count count="0"></table-count><equation-count count="0"></equation-count><ref-count count="90"></ref-count><page-count count="18"></page-count><word-count count="11332"></word-count></counts><custom-meta-group><custom-meta><meta-name>cover-date</meta-name><meta-value>January/February 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>INTRODUCTION</title><p>Coronaviruses (CoVs) are enveloped RNA viruses causing endemic infections in several mammalian and avian species. There are seven known human CoVs, each causing respiratory disease, together accounting for about one-third of common colds (<xref rid="B1" ref-type="bibr">1</xref>, <xref rid="B2" ref-type="bibr">2</xref>). Those human CoVs that have only recently entered into humans from infected animals, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and now 2019-nCoV, can cause severe, often fatal acute respiratory syndromes (<xref rid="B3" ref-type="bibr">3</xref><xref ref-type="bibr" rid="B4">–</xref><xref rid="B10" ref-type="bibr">10</xref>). CoV prevalence, facile zoonotic transmission, and potential to cause severe respiratory disease bring urgency to research aimed at discovering CoV infection mechanisms.</p><p>The mechanisms by which CoVs enter host cells, and subsequently spread intercellularly, may explain, in part, the remarkable expansion of these viruses into new hosts, including humans. Entry and spread are carried out by spike (S) glycoproteins (<xref rid="B11" ref-type="bibr">11</xref>, <xref rid="B12" ref-type="bibr">12</xref>), homotrimeric, multidomain, integral membrane glycoproteins that protrude from virions and infected cells. The S proteins adhere viruses and infected cells to host cell receptors, and they subsequently function as catalysts of virus-cell membrane fusion, and in several CoV infection settings, cell-cell membrane fusion as well. With respect to the adherence step, it is notable that many CoV S proteins contain multiple distinct host cell receptor-binding domains (RBDs) (<xref rid="B13" ref-type="bibr">13</xref><xref ref-type="bibr" rid="B14">–</xref><xref rid="B18" ref-type="bibr">18</xref>). More than one RBD raises the question of whether RBDs operate independently, and if so, whether one RBD provides complete infection competence, while the other remains as an inactive, i.e., vestigial, domain. The MERS-CoV S proteins contain a N-terminal RBD (also known as the “S1A” domain [<xref rid="B17" ref-type="bibr">17</xref>]) that is structurally similar to galectins (<xref rid="B17" ref-type="bibr">17</xref>, <xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B20" ref-type="bibr">20</xref>) and binds host cell sialic acids (<xref rid="B21" ref-type="bibr">21</xref>). MERS-CoV S proteins also have a C-terminal “S1B” RBD (<xref rid="B17" ref-type="bibr">17</xref>) that binds the protein receptor human dipeptidyl peptidase 4 (hDPP4) (<xref rid="B22" ref-type="bibr">22</xref>). Here, the S1B-hDPP4 binding is demonstrably essential for virus-cell entry (<xref rid="B23" ref-type="bibr">23</xref>, <xref rid="B24" ref-type="bibr">24</xref>), while the biological significance of the S1A-sialic acid binding is currently unclear. In contrast, the murine hepatitis virus (MHV) S proteins also have galectin-like N-terminal S1A RBDs (<xref rid="B13" ref-type="bibr">13</xref>, <xref rid="B20" ref-type="bibr">20</xref>, <xref rid="B25" ref-type="bibr">25</xref>) but presumably lack sialic acid binding potential (<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B25" ref-type="bibr">25</xref>), instead binding the protein receptor mCEACAM (mouse carcinoembryonic antigen-related cell adhesion molecule) (<xref rid="B25" ref-type="bibr">25</xref>). The MHV C-terminal S1B domains have no known receptor affinities, but they are known to control the thresholds for S-mediated membrane fusion activation (<xref rid="B26" ref-type="bibr">26</xref><xref ref-type="bibr" rid="B27">–</xref><xref rid="B29" ref-type="bibr">29</xref>). The fact that these two related beta-CoVs utilize distinct RBDs and host factors raises challenges in developing general models of CoV-cell binding, membrane fusion activation, and subsequent infection.</p><p>One hypothesis is that distinct RBDs operate at different CoV infection stages. Initial infection by virus particles likely requires durable adherence of virus particles to cell receptors. At later infection stages, CoVs produce S proteins in abundance, far exceeding the amounts that are incorporated into secreted progeny virus particles. These unincorporated S proteins accumulate on infected-cell plasma membranes where, depending on the CoV strain, they mediate cell-cell fusions, i.e., syncytial developments, which rapidly expand infection. Close cell-cell contacts may obviate the need for high-affinity adherence of S proteins to adjacent, uninfected cell surfaces. Conceivably, S1A RBDs with relatively low affinity for sialic acids might be sufficient to promote syncytial expansion of infection, without requiring high-affinity S1B-protein receptor interactions. This putative role for sialic acids has not been considered. This study provides results that implicate sialic acids in facilitating CoV infection and CoV cell-cell spread, bringing insights concerning CoV dissemination in nature.</p></sec><sec sec-type="results" id="s2"><title>RESULTS</title><sec id="s2.1"><title>JHM-CoV binds sialic acids on target cells.</title><p>Although the CoV strains comprising the MHVs have evolved protein (mCEACAM) binding determinants on their galectin-like S1A domains (<xref rid="B20" ref-type="bibr">20</xref>, <xref rid="B25" ref-type="bibr">25</xref>), we considered it likely that some MHV CoV strains also retain sialic acid binding competence. This presumption came from studies of the JHM-CoV strain of MHV. JHM-CoV can infect mCEACAM knockout mice (<xref rid="B30" ref-type="bibr">30</xref>), and it can mediate cell-cell fusion of several mCEACAM-negative cell types (<xref rid="B27" ref-type="bibr">27</xref>, <xref rid="B31" ref-type="bibr">31</xref><xref ref-type="bibr" rid="B32">–</xref><xref rid="B35" ref-type="bibr">35</xref>). Among the CoVs, only the JHM-CoV strain has these documented activities that are apparently independent of a protein receptor, making it a sensible virus to use in addressing the hypothesis that sialate- and protein receptor-binding activities coexist on CoV S1A domains.</p><p>Sialic acid-binding viruses will agglutinate erythrocytes. To determine whether sialic acid binding has a critical role in JHM-CoV entry, we evaluated hemagglutination of human erythrocytes, using influenza A virus hemagglutinin (HA)-bearing pseudo particles (IAV pp) as positive controls. Because some JHM isolates can express and incorporate sialic acid-binding hemagglutinin-esterase (HE) proteins into virus particles (<xref rid="B36" ref-type="bibr">36</xref>, <xref rid="B37" ref-type="bibr">37</xref>), we selected a recombinant JHM-CoV that lacks HE expression (JHM<sub>HE−</sub>-CoV) (see <xref ref-type="supplementary-material" rid="figS1">Fig. S1A</xref> in the supplemental material), so that any observable hemagglutination could be attributed to S proteins. Intriguingly, JHM<sub>HE−</sub>-CoV virus particles agglutinated human erythrocytes, while the related A59-CoV strain did not (<xref ref-type="fig" rid="fig1">Fig. 1A</xref>), even at particle-associated S-protein input levels slightly exceeding the corresponding JHM<sub>HE−</sub>-CoV (<xref ref-type="supplementary-material" rid="figS1">Fig. S1B</xref>). These findings suggested that JHM uniquely engages host sialic acids.</p><fig id="fig1" orientation="portrait" position="float"><label>FIG 1</label><caption><p>JHM S protein facilitates virus binding to sialic acids. (A) JHM<sub>HE-</sub>-CoV, A59-CoV, or IAV pps were serially twofold diluted, placed into V-bottom wells, and incubated for 2 h at 4°C. Human adult erythrocytes were then added to a final concentration of 0.25% (vol/vol). Hemagglutination was scored after 2 to 12 h at 4°C. The experiment was performed four times. (B) A59-CoV VLPs were collected from HEK293T cells expressing viral structural genes S, E, M, and DSP<sub>1-7</sub>-N or DSP<sub>8-11</sub>-N. VLPs were purified through 20% sucrose cushions and quantified by assessing Rluc levels. (C) Western blot detection of JHM S proteins in JHM pp, JHM VLP, and authentic JHM-CoV. (D, left) Western blot detection of A59 and JHM S proteins in VLPs. (Right) VLPs in each bar contained equal Rluc activity. (E) HeLa cells were formalin fixed, rinsed, and incubated for 2 h at 4°C with VLPs bearing A59 or JHM S. Rluc input multiplicities were equal for all VLP inoculations. Media were removed, cells were lysed, and cell-associated Rluc activities were quantified. Data are presented after subtracting background (“No S”) Rluc-positive (Rluc<sup>+</sup>) VLP levels, with each data point representing averages from independent experiments (<italic>N</italic> = 8 [A59] and 7 [JHM]; <italic>n</italic> = 4 technical replicates per experiment). Error bars present standard errors (SE) from the mean. Statistically significant deviations from background (“No S”) binding were assessed by unpaired Student’s <italic>t</italic> test and are indicated as follows: ns, not significant; *, <italic>P</italic> < 0.05; **, <italic>P</italic> < 0.01; ***, <italic>P</italic> < 0.001; ****, <italic>P</italic> < 0.0001. (F) HeLa or HeLa-mCEACAM cells were fixed and treated with vehicle (PBS) or neuraminidase (NA) for 3 h at 37°C. JHM-CoV VLPs were then added for 2 h at 4°C, cell-associated Rluc activities were quantified, and data are presented after subtracting background (“No S”) Rluc<sup>+</sup> VLP levels. Error bars present standard errors (SE) from the mean. Statistically significant deviations were assessed by unpaired Student’s <italic>t</italic> test and are indicated as follows: ns, not significant; *, <italic>P</italic> < 0.05; **, <italic>P</italic> < 0.01; ***, <italic>P</italic> < 0.001; ****, <italic>P</italic> < 0.0001.</p></caption><graphic xlink:href="mBio.02764-19-f0001"></graphic></fig><supplementary-material content-type="local-data" id="figS1"><object-id pub-id-type="doi">10.1128/mBio.02764-19.1</object-id><label>FIG S1</label><p>Western blot detection of virion proteins. (A) Purified recombinant JHM-CoV particles, normalized based on viral RNA abundance, were subjected to Western immunoblotting. Virion hemagglutinin (HE) proteins were detected using mouse anti-MHV-HE (α-MHV-HE) 5A11. (B) Purified A59-CoV and JHM<sub>HE−</sub>-CoV were subjected to Western immunoblotting. Virion spike (S) proteins were detected using mouse anti-MHV-S (α-MHV-S) (10G). The relative band intensities were used to normalize viral input in the hemagglutination assays. Download <inline-supplementary-material id="fS1" mimetype="image" mime-subtype="tif" xlink:href="mBio.02764-19-sf001.tif" content-type="local-data">FIG S1, TIF file, 1.2 MB</inline-supplementary-material>.</p><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This content is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions></supplementary-material><p>To validate the presumed JHM virus spike-host cell sialic acid interaction, we developed an MHV-based virus-like particle (VLP) system, in which VLPs contained reporter proteins that can be tracked during virus-cell binding and entry (<xref ref-type="fig" rid="fig1">Fig. 1B</xref>). In this system, VLPs were produced from 293T cells cotransfected with MHV structural genes S, E, M, and N (<xref rid="B38" ref-type="bibr">38</xref><xref ref-type="bibr" rid="B39">–</xref><xref rid="B40" ref-type="bibr">40</xref>). The N genes were modified by in-frame fusion with dual-split protein (DSP) encoding sequences, the two of which complement to form <italic>Renilla</italic> luciferase (Rluc) and green fluorescent protein (GFP) reporters. This allowed for the release of VLPs that were intrinsically Rluc and GFP positive.</p><p>We noted that MHV VLPs incorporated intact JHM spike proteins at levels that were similar to authentic viruses. This was in contrast to JHM spikes that were incorporated into vesicular stomatitis virus (VSV)-based pseudoparticles, most of which were degraded (<xref ref-type="fig" rid="fig1">Fig. 1C</xref>). These findings indicated that the VLPs structurally resemble authentic virus structures, at least with respect to spike stability and particle incorporation. As spike stability and multivalent presentation are frequently required to detect sialate interactions (<xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B41" ref-type="bibr">41</xref>), we considered the VLPs to be well-suited for receptor binding studies. Additionally, VLPs are equally produced either with or without incorporated S proteins (<xref rid="B42" ref-type="bibr">42</xref>), making it possible to use VLPs in quantifying S-protein-dependent virus-cell binding processes.</p><p>VLPs, either with or without JHM or A59 S proteins, were applied to mCEACAM-negative HeLa cells at equivalent (Rluc) multiplicities. Under these conditions, the S-protein levels on the A59 VLPs exceeded those of JHM VLPs (<xref ref-type="fig" rid="fig1">Fig. 1D</xref>). Yet, in agreement with the hemagglutinating effects of the viruses, the JHM S proteins increased VLP-HeLa cell binding above background levels, while the A59 S proteins did not (<xref ref-type="fig" rid="fig1">Fig. 1E</xref>). Notably, the JHM VLPs did not bind to HeLa cells that were pretreated with neuraminidase (<xref ref-type="fig" rid="fig1">Fig. 1F</xref>, left), suggesting that the VLP binding was dependent on cellular sialic acids.</p><p>Surprisingly, VLP binding to cells was not increased by expression of the MHV protein receptor mCEACAM, and furthermore, VLPs did not bind to mCEACAM-expressing cells that had been pretreated with neuraminidase (<xref ref-type="fig" rid="fig1">Fig. 1F</xref>, right). Together, these data suggest that, during MHV-JHM infection, the initial viral binding process is mediated by S-protein−sialic acid interactions, not by the protein receptor mCEACAM.</p></sec><sec id="s2.2"><title>Virus binding to sialic acids facilitates protein receptor-mediated entry.</title><p>We next aimed to identify the biological importance of the JHM spike-sialate interaction. We reasoned that this interaction could be sufficient for viral entry or may instead be an early step preceding viral engagement with protein (mCEACAM) receptors. To assess virus entry, we utilized VLPs containing N proteins fused to only one of the DSP fragments (DSP<sub>8-11</sub>), such that VLP entry into target cells containing DSP<sub>1-7</sub> fragments could be measured by reporter complementation into active Rluc or GFP moieties (<xref ref-type="fig" rid="fig2">Fig. 2A</xref>). We compared VLP entries in HeLa and HeLa-mCEACAM cells and found that Rluc entry signals required mCEACAM presence (<xref ref-type="fig" rid="fig2">Fig. 2B</xref>). The entry signals were elevated by the presence of TMPRSS2 (<xref ref-type="fig" rid="fig2">Fig. 2B</xref>), a known viral fusion-triggering protease (<xref rid="B11" ref-type="bibr">11</xref>, <xref rid="B12" ref-type="bibr">12</xref>, <xref rid="B43" ref-type="bibr">43</xref><xref ref-type="bibr" rid="B44">–</xref><xref rid="B48" ref-type="bibr">48</xref>). This facilitating effect of TMPRSS2 supported the contention that the VLP-based entry assay faithfully reflected authentic CoV entry processes. In conjunction with VLP binding data, these findings suggest that JHM-CoV cell entry is a multistep process, with initial virus-sialate binding and subsequent mCEACAM receptor-driven entry.</p><fig id="fig2" orientation="portrait" position="float"><label>FIG 2</label><caption><p>JHM S-protein binding to sialic acids facilitates cell entry. (A) VLPs containing DSP<sub>8-11</sub>-N were collected from transfected HEK293T cells and inoculated onto target cells expressing DSP<sub>1-7</sub>. Rluc signals arise after entry of DSP<sub>8-11</sub>-N into cells and subsequent complementation with DSP<sub>1-7</sub>. (B) HeLa target cells were transfected with DSP<sub>1-7</sub>, mCEACAM, and hTMPRSS2 as indicated. Two days posttransfection, DSP<sub>8-11</sub>-VLPs were inoculated for 6 h at 37°C, then cells were lysed, and Rluc activities were measured. (C) The indicated target cells were pretreated with vehicle or neuraminidase (NA) for 2 h at 37°C, followed by inoculation with the indicated virus particles for 2 h at 4°C. Unbound particles were removed, and after 16 h at 37°C, cells were lysed and Fluc was quantified. Data are presented as percent viral entry, normalized to vehicle controls. Statistical analyses were performed as described in the legend to Fig. 1.</p></caption><graphic xlink:href="mBio.02764-19-f0002"></graphic></fig><p>We next determined whether virus binding to sialic acids elevates protein receptor-dependent virus entry. Here, authentic JHM<sub>HE−</sub>-CoV particles were bound for 2 h to DBT (murine), HeLa-mCEACAM (human), or BHK (hamster) cells, with or without prior cell exposure to neuraminidase. Infection was then measured by quantifying viral firefly luciferase (Fluc) reporter expression. The neuraminidase pretreatments decreased JHM<sub>HE−</sub>-CoV entry by 38% (DBT), 38% (HeLa-mCEACAM), and 54% (BHK) (<xref ref-type="fig" rid="fig2">Fig. 2C</xref>). After neuraminidase pretreatments, the cells were similarly resistant to IAV pp transduction, but not VSV G protein (VSV G)-containing pseudoparticle (VSVG pp) transduction (<xref ref-type="fig" rid="fig2">Fig. 2C</xref>). Partial resistance to IAV pp, a known sialic acid-requiring virus (<xref rid="B49" ref-type="bibr">49</xref><xref ref-type="bibr" rid="B50">–</xref><xref rid="B51" ref-type="bibr">51</xref>), is best explained by incomplete removal of sialic acids by neuraminidase. We infer that the partial neuraminidase resistance of CoV VLPs is similarly explained by incomplete sialic acid removal. These results support a model in which initial virus binding to sialic acids facilitates protein receptor-mediated entry.</p></sec><sec id="s2.3"><title>Sialic acid receptors promote JHM viral spread without requiring mCEACAM protein receptors.</title><p>CoV infections spread through the canonical process of progeny virus release and reinfection, and also through fusion, i.e., syncytial spread, with uninfected cells. JHM S proteins can uniquely mediate syncytia in several conditions, even when mCEACAM protein receptors are absent (<xref rid="B33" ref-type="bibr">33</xref>, <xref rid="B35" ref-type="bibr">35</xref>). To determine whether sialic acids operate independently of mCEACAM receptors in JHM cell-cell membrane fusion and syncytial development, we cultured JHM CoV-infected mouse (DBT) cells with mCEACAM-negative human (HeLa) cells that separately harbored either DSP<sub>1-7</sub> or DSP<sub>8-11</sub>, such that HeLa cell-cell fusions would permit DSP complementation into active Rluc (<xref ref-type="fig" rid="fig3">Fig. 3A</xref>). Using live-cell Rluc substrate, the development of active Rluc was measured over time, and in the presence of increasing neuraminidase doses. The results (<xref ref-type="fig" rid="fig3">Fig. 3B</xref>) revealed neuraminidase dose-dependent reductions in JHM CoV-induced syncytia, up to 7.4-fold at the highest neuraminidase dose (2 U/ml). Similar, but less pronounced reductions were observed in parallel cultures of CEACAM-positive HeLa cells (<xref ref-type="fig" rid="fig3">Fig. 3C</xref>). Of note, the neuraminidase treatments significantly reduced cell-surface sialic acids, as revealed by adherence of fluorescent wheat germ agglutinin (WGA) to treated cells (<xref ref-type="supplementary-material" rid="figS2">Fig. S2</xref>). From these findings, we conclude that sialic acids can enable a CoV S-mediated cell-cell fusion process without requiring a prototypic proteinaceous receptor.</p><fig id="fig3" orientation="portrait" position="float"><label>FIG 3</label><caption><p>JHM-CoV cell-to-cell spread depends on host sialic acids. (A) Schematic for JHM S-protein-mediated cell-cell fusion measurements. JHM-CoV-infected DBT cells (multiplicity of infection [MOI] = 10; 5 h postinfection [hpi]) were added to target HeLa or HeLa-mCEACAM cells. The target cells were cocultures of cells expressing DSP<sub>1-7</sub> and DSP<sub>8-11</sub>. JHM-CoV-infected cells fuse with multiple target cells, which enables DSP<sub>1-7</sub>−DSP<sub>8-11</sub> complementation into active Rluc. (B and C) Quantification of Rluc over time in mCEACAM-negative (B) or mCEACAM-positive (C) target cells, in the presence of the indicated NA concentrations. Means (data points), SE (error bars), and the polynomial trend lines (<italic>R</italic><sup>2</sup> > 0.9) are shown. The results are representative of two biological repeats.</p></caption><graphic xlink:href="mBio.02764-19-f0003"></graphic></fig><supplementary-material content-type="local-data" id="figS2"><object-id pub-id-type="doi">10.1128/mBio.02764-19.2</object-id><label>FIG S2</label><p>Immunofluorescence micrographs of target cells after NA treatments. HeLa or HeLa-mCEACAM cells were pretreated with NA at the indicated doses at 37°C. Following rinses, cell surface sialate levels were assessed by coincubating cells with Alexa Fluor 647-conjugated wheat germ agglutinin (WGA-Alexa647) (Molecular Probes) at 4°C. Following fixation and Hoechst staining, images were captured y using an EMCCD Cascade 2 camera and processed in Imaris 8.3.1. Images are representative of three technical repeats. Download <inline-supplementary-material id="fS2" mimetype="image" mime-subtype="tif" xlink:href="mBio.02764-19-sf002.tif" content-type="local-data">FIG S2, TIF file, 4.5 MB</inline-supplementary-material>.</p><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This content is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions></supplementary-material></sec><sec id="s2.4"><title>A JHM-CoV spike mutation increases mCEACAM-independent cell binding and membrane fusion.</title><p>To date, analyses of the multidomain CoV S proteins have revealed that the S1A domains interact with sialic acids (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B41" ref-type="bibr">41</xref>) and that most of the S1B domains bind to protein receptors (<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B52" ref-type="bibr">52</xref>, <xref rid="B53" ref-type="bibr">53</xref>). The exceptions are the MHV beta-CoVs, which have S1A domains that bind to protein (CEACAM) receptors (<xref rid="B25" ref-type="bibr">25</xref>). From these findings, we inferred that the JHM-CoV S1A domains contain a novel dual-receptor binding capability, able to bind both sialic acid and CEACAM receptors. Sialic acid-binding sites on beta-CoV S proteins were originally inferred from mutagenesis studies (<xref rid="B19" ref-type="bibr">19</xref>), and most recently, from structural resolution of S proteins in complex with sialosides (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B54" ref-type="bibr">54</xref>). Mutations in the inferred site did decrease sialoside binding (<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B41" ref-type="bibr">41</xref>), even though they are distal from the structurally resolved sialoside binding grooves (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B54" ref-type="bibr">54</xref>). Here, we noted a mutation in the original inferred site. Among a stretch of four residues important for sialate-binding activity of bovine CoV (B-CoV) S1A, JHM differed only at residue 176, where there is a Gly on a divergent loop in place of the orthologous B-CoV Glu170 (<xref ref-type="fig" rid="fig4">Fig. 4A</xref>). To evaluate the importance of this divergence to sialic acid binding, we constructed a G176E mutant JHM-CoV S protein. G176E S-protein expression, proteolytic processing, and VLP incorporation were all comparable to the wild type (WT) (<xref ref-type="fig" rid="fig4">Fig. 4B</xref>). Notably, VLPs with G176E spikes bound more tightly to HeLa cells, and G176E spike-containing VLP binding was suppressed by neuraminidase pretreatment of the cells (<xref ref-type="fig" rid="fig4">Fig. 4C</xref>). To correlate this increased cell binding with membrane fusion, we compared the syncytial potency of JHM wild-type and G176E mutant S proteins. Cell-cell fusions were measured by DSP<sub>1–7</sub>−DSP<sub>8–11</sub> complementation (<xref ref-type="fig" rid="fig4">Fig. 4D</xref>), visualized as GFP-positive syncytia (<xref ref-type="fig" rid="fig4">Fig. 4E</xref>), and quantified by measuring Rluc signals (<xref ref-type="fig" rid="fig4">Fig. 4F</xref>). Of note, when mCEACAM receptors were present, synyctia induced by wild type and mutant S proteins developed comparably, but in the absence of mCEACAM, the G176E spikes were significantly more robust in their syncytium-inducing activity (<xref ref-type="fig" rid="fig4">Fig. 4F</xref>). We concluded that a single point mutation in the JHM-CoV S1A domain increases the cell binding and membrane fusion activities of S proteins, independent of the prototype JHM protein receptor.</p><fig id="fig4" orientation="portrait" position="float"><label>FIG 4</label><caption><p>JHM S1A G176E mutation elevates target cell binding and mCEACAM-independent cell-cell fusion. (A) Structural superimposition and sequence alignment of B-CoV S1A (PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4H14" assigning-authority="pdb">4H14</ext-link>, green) and A59 S1A (PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3JCL" assigning-authority="pdb">3JCL</ext-link>, magenta) using PyMol. Side chains in stick representation are included for residues of interest. (B) Western blot detection of VLP-associated S proteins. (C) VLPs lacking S proteins, or bearing JHM (WT) or JHM (G176E) S proteins, were applied to HeLa cells at equivalent Rluc levels. Experimental procedures, data acquisition, and processing were performed as described in the Fig. 1 legend. (D) Schematic for JHM S-protein-mediated cell-cell fusion measurements. GFP and Rluc signals arise only after S-expressing effector cells fuse with target cells, which enables DSP<sub>1-7</sub>−DSP<sub>8-11</sub> complementation. (E) Live-cell images of the syncytia formed by S-expressing cells, without (left) or with (right) mCEACAM on target cells. Images were captured by an EMCCD Cascade 2 camera and processed in Imaris 8.3.1. (F) Quantification of syncytial developments. After imaging, cells were lysed and their Rluc activities werequantified. Data are presented as fold increases above background (“No S”) values. Statistical analyses were performed as described in the Fig. 1 legend.</p></caption><graphic xlink:href="mBio.02764-19-f0004"></graphic></fig></sec><sec id="s2.5"><title>MERS-CoV spikes mediate cell fusion without requiring hDPP4 protein receptors.</title><p>MERS-CoV S1B domains bind to host protein (hDPP4) receptors (<xref rid="B22" ref-type="bibr">22</xref><xref ref-type="bibr" rid="B23">–</xref><xref rid="B24" ref-type="bibr">24</xref>), and MERS-CoV S1A domains bind to host sialic acids (<xref rid="B21" ref-type="bibr">21</xref>). This raised the question of whether MERS-CoV spikes can catalyze cell fusions after the binding of their S1A domains to cells, and independently of high-affinity protein receptors, similar to the JHM-CoV spikes. To address this question, MERS-CoV S-mediated cell-cell fusions were measured by DSP<sub>1–7</sub>−DSP<sub>8–11</sub> complementation (<xref ref-type="fig" rid="fig5">Fig. 5A</xref>) and quantified by measuring Rluc signals (<xref ref-type="fig" rid="fig5">Fig. 5B</xref>). Of note, syncytial development mediated by MERS-CoV S proteins did not require hDPP4 but did require the S-protein-activating protease human TMPRSS2 (hTMPRSS2) (<xref ref-type="fig" rid="fig5">Fig. 5B</xref>). The catalytically inactive TMPRSS2(S441A) did not facilitate cell-cell fusion (<xref ref-type="fig" rid="fig5">Fig. 5B</xref>). The MERS S-mediated cell fusions contrasted with those generated by HCoV-229E S proteins, which do not bind sialic acids (<xref rid="B21" ref-type="bibr">21</xref>). 229E S-mediated fusions absolutely required target-cell human aminopeptidase N (hAPN) receptors (<xref ref-type="fig" rid="fig5">Fig. 5B</xref>). Thus, with sufficient cell-surface protease activities, the primary hDPP4 receptor was dispensable, conceivably because the secondary sialate-binding S1A RBDs can operate at a stage in the process.</p><fig id="fig5" orientation="portrait" position="float"><label>FIG 5</label><caption><p>MERS S protein facilitates cell-cell fusion in the absence of hDPP4. (A) Schematic for MERS S-protein-mediated cell-cell fusion measurements. (B) Effector cells expressing the indicated S proteins were cocultured with target cells expressing hTMPRSS2 and/or protein receptors (hDPP4 for MERS, and hAPN for 229E). The dotted line represents background Rluc signals. (C) MERS S-expressing effector cells were mixed with hTMPRSS2 or hTMPRSS2/hDPP4 target cells in the presence of soluble MERS-S1A-Fc proteins. Data acquisition and processing were performed as described in the Fig. 1 legend.</p></caption><graphic xlink:href="mBio.02764-19-f0005"></graphic></fig><p>To determine whether the MERS S1A domains operate in the cell fusion process, S1A-Fc proteins (<xref ref-type="supplementary-material" rid="figS5">Fig. S5</xref>) were added during the assays. Exogenous S1A proteins interfered with hDPP4-independent MERS S-mediated fusion but had no effect on fusion when hDPP4 was present (<xref ref-type="fig" rid="fig5">Fig. 5C</xref>). To determine whether sialic acids operate in the cell fusion process, neuraminidase was added during the assays. Exogenous neuraminidase reduced hDPP4-independent MERS-S-mediated fusion but had no effect on the cell fusion when hDPP4 was present (<xref ref-type="supplementary-material" rid="figS3">Fig. S3</xref>). These results further suggest that either of the two S domains, S1A or S1B, can tether S proteins to target cells for subsequent cell-cell fusions, with distinct host receptors for each domain.</p><supplementary-material content-type="local-data" id="figS3"><object-id pub-id-type="doi">10.1128/mBio.02764-19.3</object-id><label>FIG S3</label><p>Target cell sialic acids affect MERS S-mediated, hDPP4-independent, cell-cell fusion. (A) Schematic for MERS S-protein-mediated cell-cell fusion measurements. Rluc signals arise only after S-expressing effector cells fuse with target cells, which enables DSP<sub>1-7</sub>−DSP<sub>8-11</sub> complementation. Syncytial development was quantified by measuring Rluc signals over time. (B and C) The kinetics of syncytial developments of hDPP4-negative (B) or hDPP4-positive (C) target cells, in the presence of the indicated NA concentrations. Means (data points), SE (error bars), and the polynomial trend lines (<italic>R</italic><sup>2</sup> > 0.9) are shown. The results are representative of two biological repeats. Download <inline-supplementary-material id="fS3" mimetype="image" mime-subtype="tif" xlink:href="mBio.02764-19-sf003.tif" content-type="local-data">FIG S3, TIF file, 2.0 MB</inline-supplementary-material>.</p><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This content is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions></supplementary-material></sec><sec id="s2.6"><title>An analogous MERS-CoV spike mutation similarly increases hDPP4-independent cell binding and membrane fusion.</title><p>We next determined whether analogous changes in S1A of the related MERS-CoV also modified viral attachment. The S1A interaction with sialic acids has biological significance, as antibodies against S1A protect mice from lethal MERS-CoV infections (<xref rid="B55" ref-type="bibr">55</xref>), and S1A acquires adaptive mutations in humans (<xref rid="B56" ref-type="bibr">56</xref>) and in mouse models of human MERS-CoV lung infection (<xref rid="B57" ref-type="bibr">57</xref>, <xref rid="B58" ref-type="bibr">58</xref>). In mice, this selectivity was at Asn222 (<xref rid="B57" ref-type="bibr">57</xref>, <xref rid="B58" ref-type="bibr">58</xref>), a known glycan addition site (<xref rid="B17" ref-type="bibr">17</xref>, <xref rid="B59" ref-type="bibr">59</xref>, <xref rid="B60" ref-type="bibr">60</xref>), and a documented hypervariable residue, evident in a group of MERS-CoV-related HKU4 bat viruses but absent in the HKU5 bat virus groups (<xref ref-type="fig" rid="fig6">Fig. 6A</xref>). Remarkably, in the context of CoV S1A structure, the Asn222 position overlaps closely with B-CoV residue Glu170 and MHV-CoV residue Gly176 (<xref ref-type="fig" rid="fig6">Fig. 6B</xref>). We constructed MERS VLPs to determine whether this Asn222 change impacted cell binding. In initial tests, we constructed MERS-CoV VLPs and evaluated their sialate binding properties. Human red blood cells (RBCs) were agglutinated by MERS VLPs, while neuraminidase-treated RBCs were not (<xref ref-type="supplementary-material" rid="figS4">Fig. S4</xref>), indicating that VLPs do bind sialosides. We then constructed wild-type and N222D VLPs containing internal Rluc and compared their Rluc and S-protein levels relative to VLPs lacking S proteins (<xref ref-type="fig" rid="fig6">Fig. 6C</xref>). VLPs were applied to human lung-derived Calu3 cells, which express the human DPP4 receptor for MERS-CoV, and to mouse lung-derived LET-1 and mouse brain-derived DBT cells, neither of which express human DPP4 and thus do not bind MERS-CoV via a protein receptor. VLP binding was assessed by quantifying cell-associated Rluc. Asn222 change did not change VLP binding to human Calu3 cells but clearly enhanced VLP binding to murine DBT and LET-1 cells (<xref ref-type="fig" rid="fig6">Fig. 6D</xref>).</p><fig id="fig6" orientation="portrait" position="float"><label>FIG 6</label><caption><p>The N222D mutation on MERS-CoV elevates target cell binding and hDPP4-independent cell-cell fusion. (A) Comparison of selected MERS S sequences relative to bat MERS-like HKU4 and HKU5. MERS S residue N222, as well as hDPP4-binding loops 1 and 2, are delineated by red boxes. MERS S1 structure is from PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=5X59" assigning-authority="pdb">5X59</ext-link>. (B) Structural superimposition of B-CoV S1A (PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=4H14" assigning-authority="pdb">4H14</ext-link>, green), A59 S1A (PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3JCL" assigning-authority="pdb">3JCL</ext-link>, magenta), and MERS S1A (PDB accession no. <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=5X4R" assigning-authority="pdb">5X4R</ext-link>, yellow) using PyMol. MERS S1A residue N222 (stick representation) is located proximally to B-CoV S1A residue E170. (C) MERS VLP binding characterization. MERS-VLPs with equivalent Rluc activities (right) were processed for Western blotting to image S-protein abundance and integrity (left). (D) VLPs were added at equivalent Rluc input multiplicities to human Calu3 or murine LET-1 cells. After 2 h at 4°C, cell-associated Rluc activities were quantified, and data were presented after subtracting background (“No S”) Rluc<sup>+</sup> VLP levels. (E) Effector cells expressing the indicated S proteins were cocultured with target cells expressing hTMPRSS2 and/or hDPP4. (F) MERS S-expressing effector cells were mixed with hTMPRSS2-expressing target cells, in the presence of the indicated Fc proteins. All Fc proteins were at 10 μM concentration throughout the coculture period. Experimental procedures, data acquisition, and processing were performed as described in the Fig. 1 legend.</p></caption><graphic xlink:href="mBio.02764-19-f0006"></graphic></fig><supplementary-material content-type="local-data" id="figS4"><object-id pub-id-type="doi">10.1128/mBio.02764-19.4</object-id><label>FIG S4</label><p>MERS VLPs agglutinate human erythrocytes. (A) The indicated IAV pps or MERS VLPs were placed in V-bottom wells and incubated for 1 h at room temperature. The VLPs (No S or MERS S) were present at equivalent levels, as measured by their internal Rluc contents. Prewarmed (37°C) human adult erythrocytes, either with or without prior NA treatment, were added to a final concentration of 0.25% (vol/vol). Hemagglutination was scored after 2 to 12 h at room temperature. The experiment was performed two times. (B) MERS VLPs (No S or MERS S) were serially twofold diluted, placed into V-bottom wells, and incubated for 1 h at room temperature. Prewarmed (37°C) human adult erythrocytes were added to a final concentration of 0.25% (vol/vol). Hemagglutination was scored after 2 to 12 h at room temperature. The experiment was performed two times. Download <inline-supplementary-material id="fS4" mimetype="image" mime-subtype="tif" xlink:href="mBio.02764-19-sf004.tif" content-type="local-data">FIG S4, TIF file, 2.3 MB</inline-supplementary-material>.</p><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This content is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions></supplementary-material><p>We next considered whether this adaptation for increased cell binding had consequences in MERS S-mediated cell-cell membrane fusion, similar to the JHM-CoV G176E mutant. We found that the N222D mutant S proteins did indeed exhibit enhanced cell fusion, relative to wild-type S proteins (<xref ref-type="fig" rid="fig6">Fig. 6E</xref>), but this was only observed by eliminating hDPP4 (<xref ref-type="fig" rid="fig6">Fig. 6E</xref>), which removes the dominant S1B-protein receptor interaction and demands S1A utilization. To further probe the relationships between S1A-cell binding and cell fusion, we determined whether N222D S1A-Fc proteins would suppress cell fusion more effectively than WT S1A-Fc. Indeed, we found that exogenously added N222D S1A-Fc proteins suppressed cell fusions more potently than the corresponding wild-type S1A-Fc did (<xref ref-type="fig" rid="fig6">Fig. 6F</xref> and <xref ref-type="supplementary-material" rid="figS5">Fig. S5</xref>), further reinforcing the direct relationship between S1A binding to cells and resultant cell-cell fusion. Altogether, these findings support the hypothesis that MERS-CoV and JHM-CoV can acquire increased cell binding through S1A mutation, which consequently allows for more robust cell-cell fusion capability. Both of these CoVs can procure these cell binding and cell fusion properties through similarly localized S1A mutations.</p><supplementary-material content-type="local-data" id="figS5"><object-id pub-id-type="doi">10.1128/mBio.02764-19.5</object-id><label>FIG S5</label><p>Western blot detection of purified Fc-tagged proteins. Protein concentration was assessed using Nanodrop (Thermo Fisher), and human IgG1 (hIgG1) (Sigma) was loaded as a quantification control. Download <inline-supplementary-material id="fS5" mimetype="image" mime-subtype="tif" xlink:href="mBio.02764-19-sf005.tif" content-type="local-data">FIG S5, TIF file, 0.8 MB</inline-supplementary-material>.</p><permissions><copyright-statement>Copyright © 2020 Qing et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qing et al.</copyright-holder><license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p>This content is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International license</ext-link>.</license-p></license></permissions></supplementary-material></sec></sec><sec sec-type="discussion" id="s3"><title>DISCUSSION</title><p>Viruses frequently begin infection by attaching to cellular sialic acids (<xref rid="B61" ref-type="bibr">61</xref>). Such viruses include several CoVs, which attach to sialates via spike S1A domains (<xref rid="B11" ref-type="bibr">11</xref>, <xref rid="B12" ref-type="bibr">12</xref>), at low affinity (<xref rid="B18" ref-type="bibr">18</xref>), but relatively high multivalent avidity (<xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B41" ref-type="bibr">41</xref>). Atomic resolution structures of 9-O-acetylated sialic acid in complex with the human OC43-CoV S1A domain (<xref rid="B18" ref-type="bibr">18</xref>) as well as several sialosides in complex with the MERS-CoV S1A (<xref rid="B54" ref-type="bibr">54</xref>) have revealed architectures of CoV-sialate binding sites. Yet even with this detailed understanding, it is not clear whether sialic acids confer susceptibility to CoV infection on their own or whether proteinaceous CoV receptors are also required. In considering this question, we first focused on the murine MHV JHM-CoV strain. This strain can infect cells and mice that lack the proteinaceous MHV receptor, mCEACAM1a (<xref rid="B27" ref-type="bibr">27</xref>, <xref rid="B30" ref-type="bibr">30</xref>, <xref rid="B32" ref-type="bibr">32</xref><xref ref-type="bibr" rid="B33">–</xref><xref rid="B35" ref-type="bibr">35</xref>, <xref rid="B62" ref-type="bibr">62</xref>, <xref rid="B63" ref-type="bibr">63</xref>). This strain is also unusually sensitive to proteolytic activation of spike-mediated membrane fusion (<xref rid="B46" ref-type="bibr">46</xref>), and therefore, we hypothesized that a low-affinity cell binding event, conceivably to cellular sialoglycans, might be sufficient for subsequent JHM-CoV protease-triggered fusion activation and cell entry.</p><p>We found that JHM-CoV binding to cells depended on host sialic acids. This binding accounted for initial virus attachment to cells, which facilitated subsequent virus engagement with proteinaceous CEACAM receptors. Later in the infection cycle, JHM-CoV spikes facilitated cell fusions and further virus dissemination in a sialic acid-dependent manner. MERS-CoV spike proteins could similarly fuse cells together without requiring prototype protein (hDPP4) receptors, presumably by utilizing sialoside receptors to engage neighboring cells. These findings, summarized in <xref ref-type="fig" rid="fig7">Fig. 7</xref>, suggest that CoV-cell entry and intercellular spread involve the lectin-like activities of spike proteins during both virus-cell attachment and infected-cell expansion into syncytia.</p><fig id="fig7" orientation="portrait" position="float"><label>FIG 7</label><caption><p>Sialic acid and protein receptor binding events during CoV infection. In the Entry panel, CoV S proteins mediate weak interactions with abundant host surface sialates, keeping viruses concentrated on cells yet potentially diffusible across plasma membranes. S proteins subsequently engage protein receptors and are proteolytically activated into membrane fusion-inducing conformations. In the Spread panel, canonical virus release is concomitant with cell-cell fusion. Cell-cell fusion involves S binding to sialic acids and does not require protein receptors, allowing infection to spread beyond the restricted distributions of protein receptors.</p></caption><graphic xlink:href="mBio.02764-19-f0007"></graphic></fig><p>While sialic acids affected virus-cell binding, infection by JHM-CoV and cell entry by MERS-CoV VLPs required the proteinaceous receptors mCEACAM and hDPP4, respectively. Therefore, one possibility is that multivalent CoV-sialate interactions are not sufficient to hold viruses throughout a virus particle-cell membrane fusion process and that sialic acids may instead only tether viruses, transiently, onto host cell surfaces, as they diffuse two-dimensionally and then bind more stably to high-affinity protein receptors. Yet, while insufficient for the initial infection, sialic acid binding may advance the spike-mediated cell-cell membrane fusions that are observed in several CoV infection processes. In cell-cell fusion, spikes on infected-cell surfaces are steadily directed at uninfected (target) cells. They are not liable to diffuse away as cell-bound viruses would, yet they still may require attachment to target cell sialates in order to effect membrane fusion. In support of this contention, we found correlations between sialic acid abundance and membrane fusion activity. This was observed for both JHM- and MERS-CoV spikes, and notably, both the JHM- and MERS-CoV spike-directed cell fusions took place independently of any proteinaceous receptors. These correlations suggest that sialic acids on uninfected cells can arrange spikes such that they will then operate cooperatively to pull opposing plasma membranes into coalescence.</p><p>This differential utilization of sialoside and protein receptors raises questions about CoV evolution. The CoV S1A domains fold into a galectin-like structure (<xref rid="B14" ref-type="bibr">14</xref>, <xref rid="B15" ref-type="bibr">15</xref>, <xref rid="B17" ref-type="bibr">17</xref><xref ref-type="bibr" rid="B18">–</xref><xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B25" ref-type="bibr">25</xref>, <xref rid="B64" ref-type="bibr">64</xref>), and several CoV S1A domains demonstrably bind carbohydrate ligands (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B41" ref-type="bibr">41</xref>, <xref rid="B54" ref-type="bibr">54</xref>), but prior to this report, the MHV-CoV S1A domains were considered to bind only protein (mCEACAM) receptors (<xref rid="B25" ref-type="bibr">25</xref>). However, Peng et al. (<xref rid="B19" ref-type="bibr">19</xref>) proposed that ancestral MHV-CoVs bound carbohydrates, with adaptive evolution generating the present-day CEACAM-binding sites. This proposal infers the existence, past or present, of evolutionary intermediates capable of binding both mCEACAM and sialic acid. The MHV JHM-CoV strain meets the criteria for such an intermediate—a virus in which a single domain retains two “receptor” binding activities, one for carbohydrate and the other for protein. Conceivably, these two receptor sites interact, such that increased affinity for one ligand reduces affinity for the other. Mutants with relatively high affinity for sialic acid may bind poorly to mCEACAM, while strains lacking sialic acid binding may demonstrate strong mCEACAM affinity. An inverse relationship would be consistent with the study of Peng et al., who proposed that acquisition of a CEACAM-binding site concomitantly destroyed a sialate site (<xref rid="B19" ref-type="bibr">19</xref>). Yet perhaps more intriguing relationships are between CoV S and hemagglutinin-esterase (HE) proteins, both of which bind sialosides (<xref rid="B65" ref-type="bibr">65</xref>, <xref rid="B66" ref-type="bibr">66</xref>). Esterase activities within HE proteins deacetylate sialosides, and it has been documented, in HKU1-CoV infections, that HE activity destroys an S-specific sialoside receptor, keeping viruses detached from cells and allowing for virus dissemination (<xref rid="B67" ref-type="bibr">67</xref>). Several MHV strains, including JHM, express a viral HE, which binds and deacetylates 4-O-acetyl sialiate (<xref rid="B68" ref-type="bibr">68</xref>). Similar to HKU-1-CoV HE, MHV HE could destroy MHV sialoside receptors and facilitate dissemination. MHV strain A59 did not bind sialic acids, and it does not retain an intact HE open reading frame (<xref rid="B36" ref-type="bibr">36</xref>, <xref rid="B37" ref-type="bibr">37</xref>), while strain JHM did bind sialic acids and does express HE, suggesting that HE is necessary for those CoVs with relatively high S-protein affinity for sialosides. MERS-CoV binds sialic acids, albeit weakly (<xref rid="B21" ref-type="bibr">21</xref>), and increases in its affinity for sialates may be limited by the absence of a MERS-CoV HE gene.</p><p>This study revealed S-protein mutations that may endow CoVs with expanded tropisms, beyond that determined by prototype proteinaceous CoV receptors. The JHM-CoV mutation G176E, engineered to reflect sialate-utilizing B-CoV, increased viral S-protein binding to cells, as well as S-protein-mediated membrane fusion, independently of mCEACAM. The MERS-CoV mutation N222D, an adaptation for virus growth in mouse lungs (<xref rid="B58" ref-type="bibr">58</xref>), operated remarkably similar to the JHM-CoV G176E change, with mutant S proteins showing increased hDPP4-independent cell binding and cell fusion. Our findings fit with the hypothesis that viruses with these mutations bind relatively tightly to sialoside receptors. However, it is clear that the changes are not present in currently identified sialoside-binding sites (<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B54" ref-type="bibr">54</xref>), which raises alternative hypotheses that include mutation-induced allosteric restructuring of sialoside-binding sites. Alternatively, the mutations may restructure distinct receptor- or coreceptor-binding sites, such as presumed binding sites for orthologous CEACAMs on MHV S proteins (<xref rid="B69" ref-type="bibr">69</xref>), or sites for CEACAM5 (<xref rid="B70" ref-type="bibr">70</xref>) or GRP78 (<xref rid="B71" ref-type="bibr">71</xref>) on MERS-CoV S proteins.</p><p>Finally, this report illuminates understanding of CoV pathogenesis. Among the CoVs, the JHM-CoV strain is known for causing lethal brain infection, even in mice that lack the principal MHV receptor, CEACAM1a (<xref rid="B30" ref-type="bibr">30</xref>). JHM spike was identified to be the major contributor to this phenotype (<xref rid="B30" ref-type="bibr">30</xref>), and JHM-CoV, but not the related A59-CoV, spread interneuronally both <italic>in vivo</italic> and within <italic>in vitro</italic> cultures of central nervous system (CNS)-derived cells (<xref rid="B89" ref-type="bibr">89</xref>, <xref rid="B90" ref-type="bibr">90</xref>). Notably, neural cell membranes are known for their abundant sialic acid content (<xref rid="B72" ref-type="bibr">72</xref>). These findings, combined with evidence that cell-to-cell syncytial spread correlates with pathogenesis in several infection models (<xref rid="B30" ref-type="bibr">30</xref>, <xref rid="B62" ref-type="bibr">62</xref>, <xref rid="B63" ref-type="bibr">63</xref>, <xref rid="B73" ref-type="bibr">73</xref><xref ref-type="bibr" rid="B74">–</xref><xref rid="B75" ref-type="bibr">75</xref>), prompts a hypothesis that JHM-CoV sialic acid binding potential accounts for an interneuronal syncytial spread that is rapidly lethal. A prediction is that variants of JHM-CoV exhibiting enhanced sialic acid affinity will have unusually high neurovirulence. Similarly, the MERS-CoV strain causes lethal pneumonia, and here it is significant that antibodies specific for the MERS-CoV S1A domains both neutralize the virus and reduce infection and pathogenesis in a mouse MERS-CoV model system (<xref rid="B55" ref-type="bibr">55</xref>, <xref rid="B59" ref-type="bibr">59</xref>, <xref rid="B76" ref-type="bibr">76</xref>). Conceivably, these antibodies interfere with sialic acid binding, reducing expansion of MERS-CoV that may take place via cell-cell fusion. Variants of MERS-CoV with enhanced cell binding may be useful in assessing the <italic>in vivo</italic> significance of the findings presented in this report.</p></sec><sec sec-type="materials|methods" id="s4"><title>MATERIALS AND METHODS</title><sec id="s4.1"><title>Cells.</title><p>HEK293T (ATCC), HeLa (ATCC), and HeLa-mCEACAM (<xref rid="B77" ref-type="bibr">77</xref>, <xref rid="B78" ref-type="bibr">78</xref>) cells were maintained in DMEM−10% FBS medium (Dulbecco’s modified Eagle medium [DMEM] containing 10 mM HEPES, 100 nM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin G, and 100 μg/ml streptomycin, and supplemented with 10% fetal bovine serum [FBS] [Atlanta Biologicals]). BHK-21 cells (ATCC) were maintained in DMEM−5% FBS medium. LET-1 cells (BEI Resources) (<xref rid="B79" ref-type="bibr">79</xref>) were maintained in DMEM−10% FBS medium lacking HEPES, sodium pyruvate and nonessential amino acids. Calu3 cells (ATCC) were maintained in MEM−20% FBS medium (minimum essential medium [MEM] supplemented with 20% FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin). DBT cells (<xref rid="B80" ref-type="bibr">80</xref>, <xref rid="B81" ref-type="bibr">81</xref>) were maintained in MEM−5% FBS medium (MEM supplemented with 5% FBS, 10% tryptose-phosphate broth, 26.8 mM sodium bicarbonate, 2 mM <sc>l-</sc>glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin). All cell lines were cultured in a 5% CO<sub>2</sub> incubator at 37°C.</p></sec><sec id="s4.2"><title>Viruses.</title><p>Recombinant MHV strains JHM.SD (<xref rid="B82" ref-type="bibr">82</xref>) and A59 (<xref rid="B83" ref-type="bibr">83</xref>), both containing a firefly luciferase (Fluc) reporter between the viral E (envelope) and M (matrix) genes, were grown in DBT cells. Media were collected at 24 to 48 h postinfection. JHM<sub>HE−</sub> arose during laboratory passaging of JHM.SD.</p></sec><sec id="s4.3"><title>Virus-like particles.</title><p>CoV virus-like particles (VLPs) were constructed by cotransfection with equimolar amounts of plasmids encoding CoV S, E (envelope), M (matrix), and N (nucleocapsid). Coding sequences for A59-CoV S, E, M, and N genes are presented in GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/AY910861.1" assigning-authority="genbank">AY910861.1</ext-link>; for JHM-CoV, GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/AC_000192.1" assigning-authority="genbank">AC_000192.1</ext-link>; and for MERS-CoV (EMC 2012 strain [<xref rid="B84" ref-type="bibr">84</xref>], GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/JX869059.2" assigning-authority="genbank">JX869059.2</ext-link>, where only the S gene is codon optimized [<xref rid="B24" ref-type="bibr">24</xref>]). The A59/JHM-CoV and the MERS-CoV genes were inserted into pCAGGS and pcDNA3.1 expression vector plasmids, respectively. Recombinant pCAGGS-DSP<sub>1-7</sub>-N and pCAGGS-DSP<sub>8-11</sub>-N were constructed by fusing the DSP<sub>1-7</sub> or DSP<sub>8-11</sub> coding sequences (pDSP<sub>1-7</sub> and pDSP<sub>8-11</sub> [<xref rid="B85" ref-type="bibr">85</xref>, <xref rid="B86" ref-type="bibr">86</xref>] provided by Zene Matsuda [University of Tokyo]), followed by a 2× SGGS linker, to the 5′ ends of the coding sequences for the N genes.</p><p>Expression plasmids (1 μg) were complexed with polyethylenimine (PEI) (Polysciences) (6 μg) in 0.2 ml of Opti-MEM (Life Technologies) for 15 min at room temperature and then added dropwise to 5 × 10<sup>5</sup> HEK293T cells in 1 ml of DMEM−10% FBS. For spikeless VLPs, S expression plasmids were replaced with empty vector plasmid DNAs. Six hours later, the cells were replenished with fresh DMEM−10% FBS. Media were collected at 48 h posttransfection.</p></sec><sec id="s4.4"><title>Pseudoviruses.</title><p>Pseudovirus <underline>p</underline>articles (pps) were constructed from a vesicular stomatitis virus (VSV) platform as described in reference <xref rid="B87" ref-type="bibr">87</xref>. Briefly, HEK293T cells were transfected with viral glycoprotein expression plasmids, including pHEF-VSV G-Indiana (BEI Resources), pcDNA3.1-HA5-QH, and pcDNA3.1-PR8 NA1 (obtained from Lijun Rong, University of Illinois—Chicago). Twenty-four hours later, the cells were inoculated for 2 h with VSVdeltaG/Junin GP-luciferase (<xref rid="B87" ref-type="bibr">87</xref>, <xref rid="B88" ref-type="bibr">88</xref>). The cells were rinsed twice with FBS-free DMEM medium and replenished with DMEM−10% FBS. Media were collected after a 48-h incubation period.</p></sec><sec id="s4.5"><title>Particle concentration.</title><p>Media containing viruses, pseudovirus particles, and VLPs were clarified by differential centrifugation (300 × <italic>g</italic>, 4°C, 10 min; 3,000 × <italic>g</italic>, 4°C, 10 min). Particles were concentrated from clarified media by overnight centrifugation (SW28, 6500 rpm, 4°C, 18 h) through a cushion comprised of 20% (wt/wt) sucrose in FBS-free DMEM. The resulting pellets were resuspended in FBS-free DMEM, and the resulting 100× concentrated particle stocks were stored at –80°C.</p></sec><sec id="s4.6"><title>Hemagglutination assay.</title><p>Serial twofold dilutions of 100× concentrated virus and pseudovirus particle stocks were prepared using FBS-free DMEM as diluent. Diluted samples were placed into V-bottom 96-well plates and incubated for 2 h at 4°C. Washed adult human erythrocytes (0.5%) (in phosphate-buffered saline [PBS]) were then added, and scoring was performed after 2- to 12-h incubation at 4°C.</p></sec><sec id="s4.7"><title>Neuraminidase treatments.</title><p>Adherent, confluent cells in 24-well plates were rinsed once with PBS before fixation with 3.7% paraformaldehyde in 0.159 M piperazine-<italic>N</italic>,<italic>N</italic>′-bis(2-ethanesulfonic acid) (PIPES) for 30 min at room temperature. Following three PBS rinses, cells were treated with vehicle or neuraminidase (1 U/ml) (catalog no. N2876; Sigma) in neuraminidase buffer (200 Mm sodium acetate [NaOAc], 2 mM CaCl<sub>2</sub> [pH 5.0]) for 3 h at 37°C. The cells were then rinsed three times with PBS and then blocked with PBS containing 2% FCS (PBS + 2% FCS) for 30 min at room temperature, and then used in virus binding assays.</p><p>For live-cell assays, adherent, confluent cells in 24- or 96-well plates were pretreated with vehicle or neuraminidase (1 U/ml) (catalog no. N2876; Sigma) in FBS-free DMEM for 2 h at 37°C. The cells were rinsed three times with PBS and then used in virus transduction assays.</p></sec><sec id="s4.8"><title>VLP binding assay.</title><p>VLPs used for binding assays were prepared by cotransfection with plasmids encoding CoV S, E (envelope), M (matrix), and DSP<sub>1-7</sub>-N + DSP<sub>8-11</sub>-N. The resulting VLPs contained complemented DSP Rluc-positive interiors. To quantify VLP-associated Rluc, VLPs were serially diluted into passive lysis buffer (PLB) (catalog no. E194A; Promega) and introduced into opaque microplate wells (50 μl per well). <italic>Renilla</italic> luciferase substrate (1.1 M NaCl, 2.2 mM Na<sub>2</sub>EDTA, 0.22 M KH<sub>2</sub>PO<sub>4</sub>, 1.3 mM NaN<sub>3</sub>, 0.44 mg/ml bovine serum albumin, 2.5 μM coelenterazine [pH 5]) was added (100 μl per well), and luminescence was read using a Veritas microplate luminometer (Turner BioSystems, Sunnyvale, CA).</p><p>VLPs were incubated for 2 h at 4°C to adherent, confluent cells, either with or without prior neuraminidase treatments. Incubations were in FBS-free DMEM and at known Rluc VLP/cell ratios. After incubation, unbound VLPs were removed, and cells were rinsed variably with PBS. To measure cell-bound VLPs, adherent cells were dissolved into PLB, and Rluc activity was quantified.</p></sec><sec id="s4.9"><title>VLP entry assay.</title><p>VLPs used for entry assays were prepared by cotransfection with plasmids encoding CoV S, E (envelope), M (matrix), and DSP<sub>8-11</sub>-N. The resulting VLPs contained DSP fragments in their interiors. To quantify VLP-associated DSP levels, VLPs were mixed with excess DSP<sub>1-7</sub>, in passive lysis buffer, for 30 min at 37°C, to allow for DSP complementation. The excess DSP<sub>1-7</sub> was obtained from HEK293 cells overexpressing these fragments. After postlysis complementation, samples were introduced into opaque microplate wells and luminescence readings were used to infer DSP<sub>8-11</sub> levels.</p><p>VLPs in FBS-free DMEM were inoculated onto target cells that were transfected 2 days earlier with pDSP<sub>1-7</sub>. Indicated experiments included cotransfection of target cells with pDSP<sub>1-7</sub> and pCAGGS-TMPRSS2<sub>FLAG</sub> (<xref rid="B44" ref-type="bibr">44</xref>). For VLP inoculations, input multiplicities were normalized to VLP DSP<sub>8-11</sub> levels. After 6 h at 37°C, the cells were rinsed three times with PBS and dissolved in passive lysis buffer, and Rluc activities were quantified.</p></sec><sec id="s4.10"><title>Virus and pseudovirus particle entry assays.</title><p>Virus particles were incubated with cells, either with or without prior neuraminidase treatments, for 2 h at 4°C. Cells were then rinsed three times with PBS and replenished with FBS-containing DMEM/MEM. After 16 h at 37°C, cells were dissolved in lysis buffer (25 mM Tris-phosphate [pH 7.8], 2 mM dithiothreitol [DTT], 2 mM 1,2-diaminocyclohexane-<italic>N</italic>,<italic>N</italic>,<italic>N</italic>′-tetraacetic acid, 10% [vol/vol] glycerol, 1% Triton X-100), and Fluc levels were quantified with Fluc substrate (1 mM <sc>d</sc>-luciferin, 3 mM ATP, 15 mM MgSO<sub>4</sub>·H<sub>2</sub>O, 30 mM HEPES [pH 7.8]) and a Veritas microplate luminometer.</p></sec><sec id="s4.11"><title>Cell-cell fusion assay.</title><p>Effector and target cells were prepared by introducing expression plasmids, using the PEI transfection method. Effector cells were cotransfected with pDSP<sub>1-7</sub> and the indicated S-expressing plasmids. S-expressing plasmids included pcDNA3.1-229E S<sub>C9</sub> (GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/AB691763.1" assigning-authority="genbank">AB691763.1</ext-link>, obtained from Fang Li, University of Minnesota). Control effector cells received pDSP<sub>1-7</sub> and empty vector plasmids. Target cells were cotransfected with pDSP<sub>8-11</sub> and the indicated S receptor-expressing plasmids. These plasmids included pCMV6-Entry-hDPP4<sub>FLAG</sub> (GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/NM_001935" assigning-authority="genbank">NM_001935</ext-link>; purchased from OriGene) and pcDNA3.1-hAPN (GenBank accession no. <ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/nuccore/M22324" assigning-authority="genbank">M22324</ext-link>; obtained from Fang Li). Indicated experiments included additional cotransfection of target cells with pCAGGS-TMPRSS2<sub>FLAG</sub> (<xref rid="B44" ref-type="bibr">44</xref>).</p><p>At 6 h posttransfection, effector and target cells were rinsed with PBS, lifted with 0.05% trypsin, and mixed at 1:1 ratios. The cocultures were incubated for 2 to 18 h at 37°C. Fused cells were visualized microscopically as green fluorescent protein-positive (GFP<sup>+</sup>) cells, and extents of cell-cell fusion were quantified as Rluc activities present in PLB cell lysates. Samples were chilled and maintained at 4°C during Rluc quantifications to eliminate DSP postlysis complementation. In time course experiments, EnduRen substrate (Promega E6482) was used in place of coelenterazine.</p></sec><sec id="s4.12"><title>Fc constructs.</title><p>pCEP4-mCEACAM-Fc was constructed previously (<xref rid="B31" ref-type="bibr">31</xref>). Splice overlap extension PCR was used to insert an MreI restriction site and GSGGGG linker codons between the mCEACAM (codons 1 to 142) and the human IgG1 splice donor site. Using this modified construct, the mCEACAM coding region was removed and replaced with MERS-S1A (codons 1 to 357), MERS S1A (N222D), and MERS S1B (codons 1 to 24 from human CD5 [hCD5] signal, followed by MERS S codons 358 to 588).</p><p>HEK293T cells were transfected using the PEI method and then incubated in FBS-free DMEM containing 2% (wt/vol) Cell Boost 5 (HyClone). Supernatants were collected on days 4 and 7 and clarified by sequential centrifugation (300 × <italic>g</italic>, 4°C, 10 min; 4,500 × <italic>g</italic>, 4°C, 10 min). The Fc-tagged proteins were then purified using HiTrap protein A high-performance columns (GE Healthcare) according to the manufacturer’s instruction. The resulting purified proteins were quantified spectrophotometrically and stored at –20°C until use.</p></sec><sec id="s4.13"><title>Western blotting.</title><p>Proteins in sodium dodecyl sulfate (SDS) solubilizer (0.0625 M Tris·HCl [pH 6.8], 10% glycerol, 0.01% bromophenol blue, 2% [wt/vol] SDS, with 2% 2-mercaptoethanol] were heated at 95°C for 5 min, electrophoresed through 8% (wt/vol) polyacrylamide-SDS gels, transferred to nitrocellulose membranes (Bio-Rad), and incubated with mouse monoclonal anti-MHV-HR2 10G (obtained from Fumihiro Taguchi, Nippon Veterinary and Life Sciences, Tokyo, Japan), mouse anti-C9 (EMD Millipore), mouse monoclonal anti-MHV HE, clone 5A11, or goat anti-human IgG (sc-2453; Santa Cruz Biotechnologies). After incubation with appropriate horseradish peroxidase (HRP)-tagged secondary antibodies and chemiluminescent substrate (Thermo Fisher), the blots were imaged and processed with a FluorChem E (Protein Simple).</p></sec><sec id="s4.14"><title>Microscopy and image acquisition.</title><p>Live-cell images were captured in a z series on an electron-multiplied charge-coupled-device digital camera (EMCCD Cascade 2; Photometrics) and deconvolved using SoftWoRx. Identical conditions were applied to all acquisitions. Deconvolved images were analyzed by an identical algorithm in Imaris 8.3.1 (Bitplane).</p></sec><sec id="s4.15"><title>Statistical analysis.</title><p>Unless stated otherwise, all experiments were repeated independently at least three times. Each data point graphed represents the mean of an independent repeat (<italic>N</italic>), generated from three or four technical repetitions (<italic>n</italic> = 3 or 4). Statistical comparisons were made by unpaired Student’s <italic>t</italic> test. Error bars indicate the standard errors of the data. <italic>P</italic> values less than 0.05 were considered statistically significant.</p></sec></sec></body><back><fn-group><fn fn-type="other"><p><bold>Citation</bold> Qing E, Hantak M, Perlman S, Gallagher T. 2020. Distinct roles for sialoside and protein receptors in coronavirus infection. mBio 11:e02764-19. <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1128/mBio.02764-19">https://doi.org/10.1128/mBio.02764-19</ext-link>.</p></fn></fn-group><ack><title>ACKNOWLEDGMENTS</title><p>This research was supported by the National Institutes of Health (NIH) under award P01 AI060699.</p><p>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</p><p>We thank Edward Campbell (Loyola University Chicago) for assistance with fluorescent confocal microscopy. 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