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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Palmitoylation of the <italic>Alphacoronavirus</italic> TGEV spike protein S is essential for incorporation into virus-like particles but dispensable for S–M interaction</title><author><name sortKey="Gelhaus, Sandra" sort="Gelhaus, Sandra" uniqKey="Gelhaus S" first="Sandra" last="Gelhaus">Sandra Gelhaus</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Thaa, Bastian" sort="Thaa, Bastian" uniqKey="Thaa B" first="Bastian" last="Thaa">Bastian Thaa</name><affiliation><nlm:aff id="aff0010">Institute for Virology, Veterinary Faculty, Free University, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany</nlm:aff></affiliation></author><author><name sortKey="Eschke, Kathrin" sort="Eschke, Kathrin" uniqKey="Eschke K" first="Kathrin" last="Eschke">Kathrin Eschke</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Veit, Michael" sort="Veit, Michael" uniqKey="Veit M" first="Michael" last="Veit">Michael Veit</name><affiliation><nlm:aff id="aff0010">Institute for Virology, Veterinary Faculty, Free University, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany</nlm:aff></affiliation></author><author><name sortKey="Schwegmann We Els, Christel" sort="Schwegmann We Els, Christel" uniqKey="Schwegmann We Els C" first="Christel" last="Schwegmann-We Els">Christel Schwegmann-We Els</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25113909</idno><idno type="pmc">7112097</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112097</idno><idno type="RBID">PMC:7112097</idno><idno type="doi">10.1016/j.virol.2014.07.035</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">001572</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001572</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Palmitoylation of the <italic>Alphacoronavirus</italic> TGEV spike protein S is essential for incorporation into virus-like particles but dispensable for S–M interaction</title><author><name sortKey="Gelhaus, Sandra" sort="Gelhaus, Sandra" uniqKey="Gelhaus S" first="Sandra" last="Gelhaus">Sandra Gelhaus</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Thaa, Bastian" sort="Thaa, Bastian" uniqKey="Thaa B" first="Bastian" last="Thaa">Bastian Thaa</name><affiliation><nlm:aff id="aff0010">Institute for Virology, Veterinary Faculty, Free University, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany</nlm:aff></affiliation></author><author><name sortKey="Eschke, Kathrin" sort="Eschke, Kathrin" uniqKey="Eschke K" first="Kathrin" last="Eschke">Kathrin Eschke</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author><author><name sortKey="Veit, Michael" sort="Veit, Michael" uniqKey="Veit M" first="Michael" last="Veit">Michael Veit</name><affiliation><nlm:aff id="aff0010">Institute for Virology, Veterinary Faculty, Free University, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany</nlm:aff></affiliation></author><author><name sortKey="Schwegmann We Els, Christel" sort="Schwegmann We Els, Christel" uniqKey="Schwegmann We Els C" first="Christel" last="Schwegmann-We Els">Christel Schwegmann-We Els</name><affiliation><nlm:aff id="aff0005">Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</nlm:aff></affiliation></author></analytic><series><title level="j">Virology</title><idno type="ISSN">0042-6822</idno><idno type="eISSN">1096-0341</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine-rich motif adjacent to the transmembrane region. This motif is palmitoylated in the <italic>Betacoronaviruses</italic> MHV and SARS-CoV. Here, we demonstrate by metabolic labeling with [<sup>3</sup>H]-palmitic acid that the S protein of transmissible gastroenteritis coronavirus (TGEV), an <italic>Alphacoronavirus</italic>, is palmitoylated as well. This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2-bromopalmitate. Mutation of individual cysteine clusters in the cysteine-rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus-like particles (VLP). Conversely, the interaction of S with the M protein, essential for VLP incorporation of S, was not impaired by lack of palmitoylation. Thus, palmitoylation of the S protein of <italic>Alphacoronaviruses</italic> is dispensable for S–M interaction, but required for the generation of progeny virions.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abrami, L" uniqKey="Abrami L">L. Abrami</name></author><author><name sortKey="Kunz, B" uniqKey="Kunz B">B. Kunz</name></author><author><name sortKey="Iacovache, I" uniqKey="Iacovache I">I. Iacovache</name></author><author><name sortKey="Van Der Goot, F G" uniqKey="Van Der Goot F">F.G. van der Goot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Abrami, L" uniqKey="Abrami L">L. Abrami</name></author><author><name sortKey="Leppla, S H" uniqKey="Leppla S">S.H. Leppla</name></author><author><name sortKey="Van Der Goot, F G" uniqKey="Van Der Goot F">F.G. van der Goot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baudoux, P" uniqKey="Baudoux P">P. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Virology</journal-id><journal-id journal-id-type="iso-abbrev">Virology</journal-id><journal-title-group><journal-title>Virology</journal-title></journal-title-group><issn pub-type="ppub">0042-6822</issn><issn pub-type="epub">1096-0341</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25113909</article-id><article-id pub-id-type="pmc">7112097</article-id><article-id pub-id-type="publisher-id">S0042-6822(14)00350-X</article-id><article-id pub-id-type="doi">10.1016/j.virol.2014.07.035</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Palmitoylation of the <italic>Alphacoronavirus</italic> TGEV spike protein S is essential for incorporation into virus-like particles but dispensable for S–M interaction</article-title></title-group><contrib-group><contrib contrib-type="author" id="au0005"><name><surname>Gelhaus</surname><given-names>Sandra</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au0010"><name><surname>Thaa</surname><given-names>Bastian</given-names></name><xref rid="aff0010" ref-type="aff">b</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au0015"><name><surname>Eschke</surname><given-names>Kathrin</given-names></name><xref rid="aff0005" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au0020"><name><surname>Veit</surname><given-names>Michael</given-names></name><xref rid="aff0010" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au0025"><name><surname>Schwegmann-Weßels</surname><given-names>Christel</given-names></name><email>christel.schwegmann@tiho-hannover.de</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005"><label>a</label>Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany</aff><aff id="aff0010"><label>b</label>Institute for Virology, Veterinary Faculty, Free University, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Tel.: +49 511 9538842; fax: +49 511 9538898. <email>christel.schwegmann@tiho-hannover.de</email></corresp><fn id="fn1"><label>1</label><p id="ntp0005">Present address: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Nobels väg 16, 171 77 Stockholm, Sweden.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>9</day><month>8</month><year>2014</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"><month>9</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>9</day><month>8</month><year>2014</year></pub-date><volume>464</volume><fpage>397</fpage><lpage>405</lpage><history><date date-type="received"><day>17</day><month>4</month><year>2014</year></date><date date-type="rev-recd"><day>18</day><month>5</month><year>2014</year></date><date date-type="accepted"><day>21</day><month>7</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><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="ab0005"><p>The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine-rich motif adjacent to the transmembrane region. This motif is palmitoylated in the <italic>Betacoronaviruses</italic> MHV and SARS-CoV. Here, we demonstrate by metabolic labeling with [<sup>3</sup>H]-palmitic acid that the S protein of transmissible gastroenteritis coronavirus (TGEV), an <italic>Alphacoronavirus</italic>, is palmitoylated as well. This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2-bromopalmitate. Mutation of individual cysteine clusters in the cysteine-rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus-like particles (VLP). Conversely, the interaction of S with the M protein, essential for VLP incorporation of S, was not impaired by lack of palmitoylation. Thus, palmitoylation of the S protein of <italic>Alphacoronaviruses</italic> is dispensable for S–M interaction, but required for the generation of progeny virions.</p></abstract><abstract abstract-type="author-highlights" id="ab0010"><title>Highlights</title><p><list list-type="simple" id="li0005"><list-item id="u0005"><label>•</label><p id="p0005">We analyzed the palmitoylation of the <italic>Alphacoronavirus</italic> TGEV spike protein.</p></list-item><list-item id="u0010"><label>•</label><p id="p0010">The cystein-rich region of the TGEV S protein is palmitoylated.</p></list-item><list-item id="u0015"><label>•</label><p id="p0015">Palmitoylation is essential for S incorporation into virus-like particles.</p></list-item><list-item id="u0020"><label>•</label><p id="p0020">Palmitoylation is dispensable for the interaction of the TGEV S with the M protein.</p></list-item></list></p></abstract><kwd-group id="keys0005"><title>Keywords</title><kwd>Coronavirus</kwd><kwd>TGEV</kwd><kwd><italic>Alphacoronavirus</italic></kwd><kwd>Spike protein</kwd><kwd>Palmitoylation</kwd><kwd>Cysteine-rich motif</kwd><kwd>Assembly</kwd></kwd-group></article-meta></front><body><sec id="s0005"><title>Introduction</title><p id="p0025">Coronaviruses (CoVs) are a family of enveloped, single-stranded, positive-sense RNA viruses that have a broad host range, as they infect a wide variety of avian and mammalian species including humans. The family <italic>Coronaviridae</italic> is split up in four genera, the <italic>Alpha</italic>-, <italic>Beta</italic>-, <italic>Delta</italic>- and <italic>Gammacoronavirus.</italic> The genus <italic>Betacoronavirus</italic> comprises the prototype coronavirus mouse hepatitis virus (MHV) as well as the recently emerged human pathogens severe acute respiratory syndrome (SARS)-CoV and middle east respiratory syndrome (MERS)-CoV. The genus <italic>Alphacoronavirus</italic> includes important animal pathogens like feline infectious peritonitis virus (FIPV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis virus (TGEV), which is the subject of this study. The positive-stranded RNA genome of coronaviruses is associated with the N protein, building up the nucleocapsid, which is surrounded by a lipid bilayer. The structural proteins Spike (S), Membrane (M), and Envelope (E) protein are embedded in this envelope.</p><p id="p0030">Coronaviruses assemble at and bud into intracellular membranes at the endoplasmic reticulum Golgi intermediate compartment, ERGIC (<xref rid="bib21" ref-type="bibr">Krijnse-Locker et al., 1994</xref>, <xref rid="bib45" ref-type="bibr">Tooze et al., 1984</xref>). Subcellular targeting signals in the viral proteins direct the proteins towards the budding site (<xref rid="bib9" ref-type="bibr">Corse and Machamer, 2002</xref>, <xref rid="bib25" ref-type="bibr">Lontok et al., 2004</xref>, <xref rid="bib33" ref-type="bibr">Paul et al., 2014</xref>, <xref rid="bib39" ref-type="bibr">Schwegmann-Wessels et al., 2004</xref>, <xref rid="bib42" ref-type="bibr">Swift and Machamer, 1991</xref>). Lateral protein interactions between M and M (<xref rid="bib24" ref-type="bibr">Locker et al., 1992</xref>), M and E (<xref rid="bib23" ref-type="bibr">Lim and Liu, 2001</xref>), M and N (<xref rid="bib29" ref-type="bibr">Narayanan et al., 2000</xref>), as well as M and S (<xref rid="bib32" ref-type="bibr">Opstelten et al., 1995</xref>) proteins play crucial roles during assembly and trigger budding. This paper focuses on the S protein of TGEV. This is a large type 1 membrane protein (250 kDa) that is responsible for the attachment to the cellular receptor and subsequent fusion of viral and cellular membranes. The S protein is incorporated into virus particles by interaction with the M protein (<xref rid="bib31" ref-type="bibr">Nguyen and Hogue, 1997</xref>, <xref rid="bib50" ref-type="bibr">Vennema et al., 1996</xref>). This is an indispensable step in the CoV replication cycle to obtain infectious virus particles.</p><p id="p0035">Palmitoylation (S-acylation) is a post-translational modification, in which a saturated fatty acid (most commonly palmitic acid) is linked to a cysteine residue by a thioester bond (<xref rid="bib38" ref-type="bibr">Schmidt et al., 1988</xref>, <xref rid="bib48" ref-type="bibr">Veit et al., 1991</xref>). This modification can have effects on protein activity (<xref rid="bib19" ref-type="bibr">Huang et al., 2010</xref>), transport (<xref rid="bib1" ref-type="bibr">Abrami et al., 2008</xref>, <xref rid="bib2" ref-type="bibr">Abrami et al., 2006</xref>), and stability (<xref rid="bib2" ref-type="bibr">Abrami et al., 2006</xref>, <xref rid="bib27" ref-type="bibr">Maeda et al., 2010</xref>). Most of the cysteines that become acylated are located near the transmembrane region (<xref rid="bib47" ref-type="bibr">Veit, 2012</xref>, <xref rid="bib53" ref-type="bibr">Yang and Compans, 1996</xref>, <xref rid="bib55" ref-type="bibr">Yik and Weigel, 2002</xref>). Palmitoylation of viral proteins has been shown to affect virus assembly or virus replication (<xref rid="bib4" ref-type="bibr">Bhattacharya et al., 2004</xref>, <xref rid="bib12" ref-type="bibr">Gaedigk-Nitschko et al., 1990</xref>).</p><p id="p0040">CoV S proteins show a conserved cysteine rich motif (CRM) in the cytoplasmic domain, adjacent to the carboxyterminal end of the transmembrane region (
<xref rid="f0005" ref-type="fig">Fig. 1</xref>). The CoV S proteins share little sequence homology at this region but the cysteine content is about 35%. For the <italic>Betacoronaviruses</italic> SARS-CoV and MHV, it has been shown that the S proteins are palmitoylated at the CRM (<xref rid="bib34" ref-type="bibr">Petit et al., 2007</xref>, <xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>). In both viruses the palmitoylation of the S protein is implicitly necessary for the incorporation of the S protein into virus-like particles (VLPs) or virus particles (<xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>, <xref rid="bib46" ref-type="bibr">Ujike et al., 2012</xref>). However, a partial CRM can be sufficient for the incorporation of the S protein (<xref rid="bib46" ref-type="bibr">Ujike et al., 2012</xref>, <xref rid="bib54" ref-type="bibr">Yang et al., 2012</xref>). In contrast to that, the interaction of the S and M proteins has different requirements in both viruses. Treatment with the general palmitoylation inhibitor 2-bromopalmitate disrupted the interaction of S and M proteins in MHV (<xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>). Conversely, the SARS-CoV S–M protein interaction is not impaired with a palmitoylation-null S protein cysteine-mutant (<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>).<fig id="f0005"><label>Fig. 1</label><caption><p>Sequence alignment of the carboxy-terminal S protein sequences from representative coronavirusspecies. Underlined cysteine residues represent potential palmitoylation sites. TGEV–PUR-46 (transmissible gastroenteritis virus, strain PUR-46), FIPV (feline infectious peritonitis virus), HCoV-NL-63 (human coronavirus NL63), SARS-CoV (severe acute respiratory syndrome coronavirus), MHV-A59 (mouse hepatitis virus, strain A59), MERS-CoV (middle east respiratory syndrome coronavirus), IBV Beaudette (infectious bronchitis virus, strain Beaudette), Bul-CoV (bulbul coronavirus).</p></caption><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0045">These reported findings demonstrate the different requirements in the closely related viruses MHV and SARS-CoV that both belong to the genus <italic>Betacoronavirus</italic>. We wanted to know whether coronaviruses from the genus <italic>Alphacoronavirus</italic> differ from these <italic>Betacoronaviruses</italic> with respect to their role of the S protein׳s CRM. In our study we addressed the role of the S protein CRM of the <italic>Alphacoronavirus</italic> TGEV during assembly. To this end, we substituted cysteines in the CRM of the TGEV S protein and assessed their effect on VLP formation and on S–M protein interaction. We show that in contrast to the SARS-CoV S protein the TGEV S protein is incorporated into VLPs irrespective of the position of its palmitoylated cysteines. For an efficient interaction between TGEV S and M proteins, palmitoylation of the S protein is not necessary. Our results demonstrate that despite general similarities, coronaviruses show differences regarding the functional role of S protein palmitoylation that may have been induced by evolutionary forces.</p></sec><sec id="s0010"><title>Results and discussion</title><sec id="s0015"><title>2-BP treatment decreases the infectivity of TGEV</title><p id="p0050">The palmitate analog 2-bromopalmitate (2-BP) inhibits protein palmitoylation in general (<xref rid="bib51" ref-type="bibr">Webb et al., 2000</xref>). To investigate whether palmitoylation has an impact on TGEV infectivity, we performed a treatment with 2-BP (
<xref rid="f0010" ref-type="fig">Fig. 2</xref>A). ST cells were infected with TGEV and treated with different concentrations of 2-BP (0, 0.8, 4, 8, and 12 µM). Vesicular stomatitis virus (VSV, grown in BHK-21 cells) was employed as a control since the assembly of this virus does not depend on palmitoylation (<xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>, <xref rid="bib52" ref-type="bibr">Whitt and Rose, 1991</xref>). These 2-BP concentrations were not cytotoxic for ST cells verified with a WST-1 cell proliferation assay (<xref rid="f0010" ref-type="fig">Fig. 2</xref>B). About 24 h after infection, media were harvested and viral infectivity was determined by plaque assay. The production of infectious VSV particles was not significantly inhibited by the 2-BP treatment (<xref rid="f0010" ref-type="fig">Fig. 2</xref>A) (<xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>, <xref rid="bib52" ref-type="bibr">Whitt and Rose, 1991</xref>). In contrast to that, a significant, dose-dependent decrease of TGEV infectivity was observed upon 2-BP treatment. This decrease in infectivity correlated with a decrease in the amount of virus particles in the supernatant of 2-BP-treated, infected cells, as seen by Western blot of pelleted supernatants for viral proteins (<xref rid="f0010" ref-type="fig">Fig. 2</xref>C). The amount of protein expression in the cell lysates was not influenced by 2-BP treatment (<xref rid="f0010" ref-type="fig">Fig. 2</xref>C). From these results, we conclude that an inhibition of palmitoylation has negative effects on the production of infectious TGEV (but not VSV) particles. While we cannot exclude that the reduction in infectious TGEV particle numbers upon 2-BP treatment is partially caused by inhibited palmitoylation of an essential cellular factor, we considered it most likely that the palmitoylation of a viral protein is required for the production of infectious virions. This led us to assess whether the TGEV S protein is palmitoylated.<fig id="f0010"><label>Fig. 2</label><caption><p>Palmitoylation of TGEV. (A) Treatment with 2-bromopalmitate (2-BP) decreases the infectivity of TGEV. ST or BHK-21 cells were inoculated with TGEV or VSV, respectively. At 1 h p.i., media were replaced with growth media containing the indicated concentrations of 2-BP or ethanol as 2-BP solvent (ethanol control: amount of ethanol present in the highest 2-BP concentration; 0.0 µM 2-BP: medium without ethanol). Twenty-four hours later, media were collected and the amount of infectious virus particles was determined by plaque assay. Plaque forming units (PFU)/ml are displayed as mean+standard deviation (SD). <sup>⁎</sup>, significantly different according to Student׳s <italic>t</italic> test compared to 0.0 µM 2-BP (<italic>p</italic><0.05). (B) 2-BP is not cytotoxic to ST cells at the concentrations used. ST cells were treated with the indicated concentrations of 2-BP and cell proliferation was determined by WST 1-assay. The cell proliferation is indicated as mean+SD in percentage. (C) The amount of viral proteins present in viral particles is reduced after 2-BP treatment. Virions from cell culture supernatants of infected cells, treated with the indicated amounts of 2-BP, were pelleted by ultracentrifugation 24 h p.i. and analyzed by SDS-PAGE and Western blotting (virions). The indicated proteins (S, N, and M) were detected by monoclonal antibodies. The corresponding cells were lysed and the respective proteins were equally analyzed (cell lysates). (D) Palmitoylation of the TGEV S protein in virus particles. Infected and mock-infected ST cells were labeled with [<sup>3</sup>H]-palmitic acid or [<sup>35</sup>S]-methionine/cysteine. Radiolabeled virus was analyzed by SDS-PAGE and fluorography. A hydroxylamine treatment was performed to determine the linkage of fatty acids via a thioester bond.</p></caption><graphic xlink:href="gr2_lrg"></graphic></fig></p></sec><sec id="s0020"><title>TGEV S is palmitoylated in virus particles</title><p id="p0055">Recent studies have shown that the S proteins of MHV and SARS-CoV, both members of the genus <italic>Betacoronavirus</italic>, are palmitoylated (<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>, <xref rid="bib34" ref-type="bibr">Petit et al., 2007</xref>, <xref rid="bib41" ref-type="bibr">Shulla and Gallagher, 2009</xref>, <xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>). The cysteine-rich motif (CRM) is highly conserved among CoV S proteins, and a CRM is also found in the S protein of TGEV as a member of the genus <italic>Alphacoronavirus</italic> (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). To determine whether the TGEV S protein is also palmitoylated, TGEV was grown in ST cells in the presence of [<sup>3</sup>H]-palmitic acid to achieve radioactive labeling, followed by pelleting of the virus, SDS-PAGE and fluorography. This revealed that the S protein of TGEV is indeed S-acylated, visible by the band at about 250 kDa which was absent in uninfected cells (<xref rid="f0010" ref-type="fig">Fig. 2</xref>D). The specificity of this labeling with [<sup>3</sup>H]-palmitic acid was confirmed by hydroxylamine treatment (<xref rid="f0010" ref-type="fig">Fig. 2</xref>D). Hydroxylamine cleaves thioester linkages at neutral pH and thus any palmitic acid linked to a cysteine residue by S-acylation. The disappearance of the TGEV S protein band after hydroxylamine treatment proves that this protein is S-acylated at at least one cysteine residue.</p></sec><sec id="s0025"><title>Palmitoylation of TGEV S is necessary for incorporation into VLPs</title><p id="p0060">As the infectivity of TGEV was reduced after 2-BP treatment and the S protein present in TGE virions was shown to be palmitoylated, we concentrated our analysis on the cysteine-rich motif (CRM) present in the cytoplasmic domain of the TGEV S protein (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). The Swt protein of the TGEV–PUR-MAD strain contains 10 cysteine residues within the CRM (<xref rid="bib37" ref-type="bibr">Sanchez et al., 1990</xref>). The CRM of TGEV S can be split up in three cysteine clusters with three to four cysteine residues (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). To test whether specific cysteine clusters have a crucial role during virus assembly, partial Cys-mutants were created in addition to the complete cysteine mutant where all cysteines were replaced by alanines (S_C<sub>1–10</sub>A,
<xref rid="f0015" ref-type="fig">Fig. 3</xref>A). As the transfection efficiency of ST cells is quite low, BHK-21 cells were transfected with the plasmids encoding for the different Cys-mutants. The labeling with [<sup>3</sup>H]-palmitic acid showed that all partial mutants were palmitoylated whereas the complete Cys-mutant was not palmitoylated (<xref rid="f0015" ref-type="fig">Fig. 3</xref>B). The presence of three cysteines in the CRM (S_C<sub>1–7</sub>A) was hence sufficient for the protein to be acylated even if the degree of palmitoylation was lower for this mutant (<xref rid="f0015" ref-type="fig">Fig. 3</xref>B). It is conceivable that palmitoylation of the CRM cysteines is important for fusion, which is mediated by the S protein. The importance of the CRM of the MHV and SARS-CoV S proteins for fusion has already been proven (<xref rid="bib5" ref-type="bibr">Bos et al., 1995</xref>, <xref rid="bib8" ref-type="bibr">Chang et al., 2000</xref>, <xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>, <xref rid="bib41" ref-type="bibr">Shulla and Gallagher, 2009</xref>). In this study, we however evaluated whether palmitoylation is important for assembly and incorporation of the spike protein into newly generated virus particles. To analyze this role a virus-like particle assay was performed in which the incorporation of S can be analyzed separately from the influence of the fusion capacity of the S protein. The incorporation of the TGEV S protein is an essential step during assembly to form infectious particles. To determine the importance of the TGEV S protein׳s CRM during virus assembly we analyzed the capability of the TGEV S Cys-mutants to be incorporated into virus-like particles (VLPs, <xref rid="f0015" ref-type="fig">Fig. 3</xref>D and E). The co-expression of the TGEV E and M proteins is the minimal requirement for VLP formation (<xref rid="bib3" ref-type="bibr">Baudoux et al., 1998</xref>). If the S protein is co-expressed with M and E it will be incorporated into VLPs (<xref rid="bib50" ref-type="bibr">Vennema et al., 1996</xref>). In contrast to the authentic TGEV S protein the complete Cys-mutant S_C<sub>1–10</sub>A was not incorporated into VLPs (<xref rid="f0015" ref-type="fig">Fig. 3</xref>D). The analyzed partial Cys-mutants S_C<sub>1–3</sub>A, S_C<sub>4–7</sub>A, S_C<sub>8–10</sub>A, and S_C<sub>1–7</sub>A behaved like the TGEV Swt protein and were incorporated into VLPs (<xref rid="f0015" ref-type="fig">Fig. 3</xref>D). To confirm these results and to account for variations in protein levels in Western blots, we additionally performed immunofluorescence analysis to show the cell surface transport of S protein (<xref rid="f0015" ref-type="fig">Fig. 3</xref>E). We obtained comparable intracellular expression levels of all tested TGEV S proteins and the TGEV M protein. Like in the VLP assay analyzed by Western blot, the S_C<sub>1–10</sub>A mutant was the only S protein which was not detectable on the cell surface and therefore not incorporated into VLPs (<xref rid="f0015" ref-type="fig">Fig. 3</xref>E). These results demonstrate that cysteines are important for the S protein incorporation into viral particles, and that they are at least partially redundant. In contrast to the reports for SARS-CoV (<xref rid="bib46" ref-type="bibr">Ujike et al., 2012</xref>), the specific position of the cysteine residue inside the cysteine-rich region seems not to be relevant for the incorporation into VLPs. The presence of one cluster of 3 cysteines (S_C<sub>1–7</sub>A) is enough to incorporate S into VLPs.<fig id="f0015"><label>Fig. 3</label><caption><p>Palmitoylation of S is necessary for incorporation into VLPs. (A) Schematic illustration of TGEV S wild-type and TGEV S cysteine-mutants. The cysteine-rich motif (CRM) and the tyrosine-based retention signal are highlighted by boxes. Cysteines substituted by alanines are written in bold. ED, ectodomain; TMD, transmembrane domain; CD, cytoplasmic domain. (B) The complete TGEV S cysteine-mutant is not palmitoylated, whereas the partial cysteine-mutants are. BHK-21 cells expressing TGEV Swt or the indicated TGEV S cysteine-mutants were labeled with [<sup>3</sup>H]-palmitic acid or [<sup>35</sup>S]-methionine/cysteine for 5 h. The cells were lysed and S protein was immunoprecipitated and subjected to SDS-PAGE and fluorography. (C) Lack of cysteines in the CRM does not affect oligomerization. BHK-21 cells expressing TGEV Swt or S_C<sub>1–10</sub>A were labeled with [<sup>35</sup>S]-methionine/cysteine for 6 h, then solubilized in 6 mM dodecyl-β-<italic>m</italic>-maltoside. A sucrose density gradient was overlaid with these cell lysates and was ultracentrifuged. Fractions were collected, immunoprecipitated, and subjected to SDS-PAGE and phosphorimaging. (D) TGEV Swt and partial TGEV S cysteine-mutants can be incorporated into VLPs, whereas a complete TGEV S cysteine-mutant cannot. BHK-21 cells were transfected with expression plasmids for the indicated versions of TGEV S, and for TGEV M and E proteins (+) or a mock expression plasmid (−). At 24 h p.t., VLPs were collected by ultracentrifugation through a sucrose cushion, and analyzed by SDS-PAGE and Western blotting. The corresponding cell lysates were checked for the presence of the respective proteins. (E) The TGEV S protein is detectable on the cell surface of BHK-21 cells after co-expression of S, M, and E proteins. BHK-21 cells were transfected with expression plasmids for the indicated versions of TGEV S alone or together with TGEV M and E encoding plasmids. At 24 h p.t., the S protein expression was detected by cell surface immunostaining or staining after permeabilization of the cells. In addition to the TGEV S protein the TGEV M protein was detected intracellularly in the parallel co-expression experiment.</p></caption><graphic xlink:href="gr3_lrg"></graphic></fig></p><p id="p0065">The S proteins of coronaviruses are known to form trimers (<xref rid="bib11" ref-type="bibr">Delmas and Laude, 1990</xref>). To exclude that the inability of the complete Cys-mutant to incorporate into VLPs is caused by a defect in oligomerization, we analyzed the quaternary structure of S_C<sub>1–10</sub>A with a sucrose density gradient and compared it with the TGEV Swt protein. The results showed that the wild type protein and the Cys-mutant are both detectable in high-concentration sucrose fractions indicating a comparable oligomerization potential (<xref rid="f0015" ref-type="fig">Fig. 3</xref>C). In conclusion, the palmitoylation of the TGEV S is necessary for the incorporation of the S protein into VLPs, but does not affect oligomerization of S.</p><p id="p0070">How does palmitoylation of the TGEV S protein influence S incorporation?</p><p id="p0075">One possible explanation might be that the conformation of the cytoplasmic tail of the S protein is crucial for the incorporation of S protein into virus particles. The palmitic acids probably help in membrane attachment of the cytoplasmic tail and may thus define its conformation. This conformation may play a minor role in the binding to the M protein but may be important for the incorporation of the TGEV S protein into virus-like particles. It is possible that the linked fatty acids determine how the protein is positioned in the ERGIC membrane and how it is inserted in the dense matrix formed by the lateral M–M interactions described for coronavirus assembly (<xref rid="bib10" ref-type="bibr">de Haan et al., 2000</xref>, <xref rid="bib30" ref-type="bibr">Neuman et al., 2006</xref>).</p><p id="p0080">The palmitoylation of proteins can concentrate them at certain membrane domains (<xref rid="bib18" ref-type="bibr">Heakal et al., 2011</xref>). A lot of viruses use palmitoylation to target their proteins to lipid microdomains at the plasma membrane for assembly (<xref rid="bib4" ref-type="bibr">Bhattacharya et al., 2004</xref>, <xref rid="bib56" ref-type="bibr">Zhang et al., 2000</xref>). Palmitoylated proteins concentrate with interacting proteins at intracellular microdomains as well. As examples, the chaperon Calnexin and the transmembrane thioredoxin protein are recruited to detergent-resistant membranes of the endoplasmic reticulum by palmitoylation (<xref rid="bib17" ref-type="bibr">Hayashi and Fujimoto, 2010</xref>, <xref rid="bib26" ref-type="bibr">Lynes et al., 2012</xref>). Thus, a version of the TGEV S protein like the complete Cys-mutant S_C<sub>1–10</sub>A that lacks palmitic acids may interact with the M protein in an S–M protein co-expression experiment but may not be sorted to the membrane microdomains at the ERGIC in which virus assembly and budding take place and may therefore not be incorporated into newly generated virions or VLPs. McBride and Machamer have shown that the SARS-CoV S protein is present in DRMs at the plasma membrane in single expression experiments. For a complete Cys-mutant of the SARS-CoV S protein it was shown that it is no longer present in DRMs (<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>). These results confirm the hypothesis that the palmitoylation of coronavirus S proteins directs them to membrane microdomains and that this localization is indispensable for the incorporation into VLPs and virions.</p></sec><sec id="s0030"><title>Palmitoylation of TGEV S is dispensable for interaction with the M protein</title><p id="p0085">Lateral protein interactions play important roles during virus assembly. The interaction of S and M proteins is essential for the incorporation of S into virus particles. Thus, we aimed to find out whether the palmitoylation of S is required for the interaction with the M protein (as in the case of MHV;(<xref rid="bib41" ref-type="bibr">Shulla and Gallagher, 2009</xref>; <xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>)), or not (as for SARS-CoV;(<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>; <xref rid="bib46" ref-type="bibr">Ujike et al., 2012</xref>)). To determine this, we expressed M (with HA-tag) together with the various cysteine mutants of S, with the background of a destroyed ERGIC retention signal (Y/A,
<xref rid="f0020" ref-type="fig">Fig. 4</xref>A). With this Y/A mutation, S is efficiently transported to the plasma membrane in the absence of M, but is retained intracellularly by M (<xref rid="f0020" ref-type="fig">Fig. 4</xref>B and C). Failure of S_Y/A cysteine mutants to interact with M thus leads to plasma membrane delivery of S even in the presence of M. The TGEV S variants depicted in <xref rid="f0020" ref-type="fig">Fig. 4</xref>A were expressed in BHK-21 cells by transfection, in the absence or presence of HA-tagged TGEV M. Immunofluorescence analysis was employed to assess the localization of S and M. All partial Cys-mutants and even the complete Cys-mutant showed a similar protein localization pattern with the M protein (<xref rid="f0020" ref-type="fig">Fig. 4</xref>B and C). This pattern was identical to the one seen for the S wildtype protein in coexpression with the HA-tagged M protein (S wt, <xref rid="f0020" ref-type="fig">Fig. 4</xref>B and C). In contrast to that, the VSV G protein was not retained and not intracellularly clustered by the co-expression of HA-tagged TGEV M protein (VSV G, <xref rid="f0020" ref-type="fig">Fig. 4</xref>B and C). These results indicate that specific interactions between the TGEV S and M proteins lead to an intracellular retention of the mutant S proteins and that the interaction of TGEV S and M proteins is not dependent on the palmitoylation of S. Palmitoylation is known to affect protein trafficking (<xref rid="bib15" ref-type="bibr">Greaves and Chamberlain, 2007</xref>, <xref rid="bib35" ref-type="bibr">Resh, 1999</xref>, <xref rid="bib36" ref-type="bibr">Resh, 2006</xref>). Our results show that this is not the case for the TGEV S since the full cysteine-mutant with Y/A-mutation traffics correctly to the cell surface in single expression (<xref rid="f0020" ref-type="fig">Fig. 4</xref>C) and the full cysteine-mutant has a comparable oligomerization potential as the wildtype protein (<xref rid="f0015" ref-type="fig">Fig. 3</xref>C).<fig id="f0020"><label>Fig. 4</label><caption><p>Palmitoylation of TGEV S is dispensable for interaction with the M protein. (A) Schematic illustration of TGEV S_Y/A and TGEV S cysteine-mutants with Y/A-mutations. The cysteine-rich motif (CRM) and the destroyed tyrosine-based retention signal are highlighted. Substitutions by alanine are written in bold. ED, ectodomain; TMD, transmembrane domain; CD, cytoplasmic domain. (B) Every TGEV S cysteine-mutant shows an intracellular clustering in co-expression with the HA-tagged TGEV M protein. BHK-21 cells expressing either the S proteins alone (mutants shown in A, S wt, or VSV G) or in co-expression with the TGEV M protein were permeabilized with Triton-X 100 prior to antibody incubation. The same field is shown in each set of images for the coexpression of S and M. (C) Every TGEV S cysteine-mutant is retained intracellularly when co-expressed with the HA-tagged TGEV M protein. The S protein (or the VSV G protein) was detected on the cell surface of non-permeabilized BHK-21 cells expressing either the S proteins alone or in co-expression with the TGEV M. The detection of the HA-tagged TGEV M protein was done after permeabilization with Triton-X 100 prior to antibody incubation. The same field is shown for the detection of surface-expressed S together with the total M staining. (D) Comparison of the role of coronaviral S protein palmitoylation for MHV (references indicated), SARS-CoV (references indicated), and TGEV (this study) for S–M protein interaction and S incorporation into VLPs or virions.</p></caption><graphic xlink:href="gr4_lrg"></graphic></fig></p><p id="p0090">Studies on MHV showed that the cytoplasmic tail of the S protein and, more specifically, the membrane adjacent cysteine-rich region is important for the interaction with M and for incorporation into virus-like particles (<xref rid="bib6" ref-type="bibr">Bosch et al., 2005</xref>, <xref rid="bib14" ref-type="bibr">Godeke et al., 2000</xref>). Our results for TGEV are similar to the published results for the SARS-CoV S protein, where the cysteine-rich motif is not important for the interaction between S and M proteins (<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>). For these coronaviral spike proteins other parts like the transmembrane domain or the ectodomain of S could play a role in the interaction with the M protein.</p></sec></sec><sec id="s0035"><title>Conclusion</title><p id="p0095">It is of significant interest to understand how the S protein is incorporated into coronavirus particles because the S protein mediates binding to the cellular receptor and fusion with the cellular membrane and is therefore indispensable for the generation of infectious virus particles. One important result from our study is that the palmitoylation of the cysteine-rich motif at the carboxy-terminus of the TGEV S protein is necessary for the incorporation of S into virus-like particles. Furthermore, our results suggest that the loss of palmitoylation does not affect the interaction with the TGEV M protein. Here, we show that for TGEV, the interaction of the S protein with M has different requirements than the incorporation of S into virus particles.</p><p id="p0100">This is the first study about the role of palmitoylation in virus assembly for the genus <italic>Alphacoronavirus</italic> in the family <italic>Coronaviridae</italic>, demonstrating that there are some similarities but also differences in the requirements for coronavirus assembly in MHV (<xref rid="bib41" ref-type="bibr">Shulla and Gallagher, 2009</xref>, <xref rid="bib44" ref-type="bibr">Thorp et al., 2006</xref>, <xref rid="bib54" ref-type="bibr">Yang et al., 2012</xref>), SARS-CoV (<xref rid="bib28" ref-type="bibr">McBride and Machamer, 2010</xref>, <xref rid="bib46" ref-type="bibr">Ujike et al., 2012</xref>), and the <italic>Alphacoronavirus</italic> TGEV (<xref rid="f0020" ref-type="fig">Fig. 4</xref>D).</p><p id="p0105">Future studies about the role of palmitoylation of the TGEV S protein in virus assembly should include the analysis of recombinant virions that contain the respective cysteine mutations. Additionally it should be analyzed if the TGEV S protein is directed to distinct membrane microdomains inside the ERGIC and if this localization is determined by cysteine palmitoylation.</p></sec><sec id="s0040"><title>Materials and methods</title><sec id="s0045"><title>Cells</title><p id="p0110">BHK-21 cells were grown in Eagle׳s minimal essential medium (EMEM; Gibco BRL) supplemented with 5% fetal calf serum (FCS; Biochrom). ST (swine testicular) cells were propagated in Dulbecco׳s minimal essential medium (DMEM; Gibco BRL) containing 10% Mycoplex fetal bovine serum (GE Healthcare).</p></sec><sec id="s0050"><title>Plasmid DNAs, construction of plasmids</title><p id="p0115">The TGEV S gene, strain PUR-46-MAD (<xref rid="bib37" ref-type="bibr">Sanchez et al., 1990</xref>), was cloned into the pCG1 plasmid using <italic>Bam</italic>HI and <italic>Pst</italic>I restriction sites (<xref rid="bib7" ref-type="bibr">Cathomen et al., 1995</xref>, <xref rid="bib40" ref-type="bibr">Schwegmann-Wessels et al., 2006</xref>). Cysteine mutants of TGEV S protein and TGEV S_Y/A protein were generated by a standard hybridization PCR technique, as described previously (<xref rid="bib39" ref-type="bibr">Schwegmann-Wessels et al., 2004</xref>). The generated cysteine mutant proteins are illustrated in <xref rid="f0015" ref-type="fig">Figs. 3</xref>A and <xref rid="f0020" ref-type="fig">4</xref>A. An HA-peptide (MYPYDVPDYA) was C-terminally fused to the TGEV M protein using standard PCR techniques. The mutated gene regions generated by PCR were verified by sequencing (Eurofins MWG Operon).</p></sec><sec id="s0055"><title>Transient transfections (PEI and Lipofectamine)</title><p id="p0120">Transfection of BHK-21 cells in 6- and 24-well-plates was performed by using Lipofectamine 2000 reagent (Life Technologies) following the manufacturer׳s instructions. BHK-21 cells in 10 cm-diameter dishes were transfected by using Polyethylenimine (PEI; Polysciences). Transfections were carried out at a confluency of 70–80%. The medium was replaced with 7 ml fresh EMEM containing 3% FCS. A total of 12 µg DNA were pipetted in 3 ml Opti-MEM (Life Technologies) and were incubated for 5 min. Then, 20 µl of a 1 µg/µl PEI-working solution was added. The mixture was incubated for 15 min at room temperature and added to the cells.</p></sec><sec id="s0060"><title>2-bromopalmitate treatment and plaque assay</title><p id="p0125">TGEV was propagated on ST cells and VSV (strain Indiana) on BHK-21 cells. Infections were carried out on confluent monolayers with an MOI of 0.001 at 37 °C on a rocking shaker. At 1 h p.i. media were removed and cells rinsed with PBS to prepare for further treatments. For 2-bromopalmitate (2-BP) treatment infected cells were grown in DMEM containing variable amounts of 2-BP (0, 0.8, 4, 8, and 12 µM) or ethanol (solvent of 2-BP). At a detectable cytopathic effect supernatants were harvested and spun at 2800<italic>g</italic> for 10 min at 4 °C to remove cellular debris. The amount of infectious virus particles was determined by plaque assay as described by <xref rid="bib20" ref-type="bibr">Krempl et al. (2000)</xref> using ST cells (TGEV) or BHK-21 cells (VSV). The cytotoxicity of the 2-BP treatment on ST cells was quantified with the cell proliferation reagent WST-1 (Roche) in 96-well microplates according to the manufacturer׳s instructions. Viral proteins (S, M, and N) incorporated into virions were detected via SDS-PAGE and Western blot analysis after ultracentrifugation of the respective cell culture supernatants. S protein was detected with mAB 6A.C3 (1:200), N protein with anti-coronavirus monoclonal antibody FIPV3-70 (1:1000, Thermo Scientific), and M protein with mAB 9D.B4 (1:200) followed by incubation with an anti-mouse peroxidase-conjugated antibody (Dako; 1:1000). Protein expression in the cells was equally detected after cell lysis.</p></sec><sec id="s0065"><title>Labeling of viral particles with [<sup>3</sup>H]-palmitic acid</title><p id="p0130">ST cells in 15 cm-diameter dishes were mock-infected or infected with TGEV at an MOI of 10. At 3 h p.i. the cells were labeled with 20 µCi/ml [<sup>3</sup>H]-palmitic acid ([9,10-<sup>3</sup>H(N)], Perkin-Elmer) or [<sup>35</sup>S]-Met/Cys (EasyTagExpress mix, Perkin-Elmer) in Met/Cys-free Dulbecco׳s modified Eagle׳s medium (DMEM; PAN Biotech) supplemented with 5 mM <sc>l</sc>-glutamine (PAN Biotech). At 24 h p.i supernatants were harvested and cleared by low-speed centrifugation (3000<italic>g</italic>, 20 min, 4 °C) followed by pelleting of the virus by ultracentrifugation in an SW28m rotor (Beckman Coulter) at 28,000 rpm for 2 h at 4 °C. The pellet was resuspended in 2× non-reducing SDS sample buffer (0.1 M Tris–HCl, 2% SDS, 20% glycerol, 2% bromophenol blue) and analyzed by SDS-PAGE and fluorography, as described previously (<xref rid="bib43" ref-type="bibr">Thaa et al., 2011</xref>). To determine an ester-type linkage of the acylated viral protein a hydroxylamine treatment under neutral pH conditions was performed, as described previously (<xref rid="bib49" ref-type="bibr">Veit et al., 2008</xref>).</p></sec><sec id="s0070"><title>Metabolic labeling with [<sup>3</sup>H]-palmitic acid and immunoprecipitation</title><p id="p0135">BHK-21 cells grown in 6-well-plates were transfected with TGEV S expression plasmids using Lipofectamine 2000. Twenty-four hours post-transfection, cells were washed with phosphate-buffered saline (PBS) and incubated for 1 h in Met/Cys-free DMEM supplemented with 5 mM <sc>l</sc>-glutamine. Subsequently, cells were labeled for 5 or 19 h with 1000 µCi/ml [<sup>3</sup>H]-palmitic acid. A parallel dish was labeled with 600 μCi/ml [<sup>35</sup>S]-Met/Cys. Cells were lysed in NP-40 lysis buffer (0.5% sodium deoxycholate, 1% Nonidet P40, 150 mM NaCl, 50 mM Tris–HCl, pH 7.5) containing protease inhibitor (Complete, Roche) for 30 min at 4 °C. Lysates were clarified for 30 min at 20,000<italic>g</italic>, 4 °C. S protein was immunoprecipitated with mouse anti-TGEV S monoclonal antibody, 6A.C3 (<xref rid="bib13" ref-type="bibr">Gebauer et al., 1991</xref>) and protein A-sepharose (Sigma) overnight at 4 °C. Immunoprecipitates were washed three times with NP-40 lysis buffer and were eluted in 2× non-reducing SDS sample buffer at 96 °C for 10 min followed by SDS-PAGE and fluorography.</p></sec><sec id="s0075"><title>Indirect immunofluorescence microscopy</title><p id="p0140">BHK-21 cells grown on coverslips in 24-well-plates were co-transfected with pCG1-TGEV-S wt, pCG1-TGEV-S_Y/A, pCG1-TGEV-Cys-mutants, pCG1-VSV-G with empty vector (pCG1) or with HA-tagged pCG1-TGEV-M. The transfection ratio of S to M was 5:1. At 18 h p.t. cells were fixed with 3% paraformaldehyde in PBS and indirect immunofluorescence analysis was performed. For intracellular protein detection cells were permeabilized with 0.2% Triton/PBS for 5 min. Detection of TGEV S protein was performed as described previously (<xref rid="bib39" ref-type="bibr">Schwegmann-Wessels et al., 2004</xref>). The VSV G protein was detected with the monoclonal antibodies I-1 and I-14 (<xref rid="bib16" ref-type="bibr">Hanika et al., 2005</xref>). As secondary antibody, a Cy3-conjugated sheep–anti-mouse antibody (1:300, Sigma) was used. The HA-tagged TGEV M was visualized by an anti-HA-tag antibody (rabbit, 1:200, Sigma) and anti-rabbit fluorescein isothiocyanate (FITC)-labeled secondary antibody (goat anti-rabbit, 1:300, Sigma). Fluorescence micrographs were acquired on a Nikon Eclipse Ti microscope. For the detection of S protein present in VLPs on the cell surface plasmids encoding for the respective S proteins were co-transfected with TGEV M and E plasmids. The immunostaining was performed as described above with and without permeabilization of the cells.</p></sec><sec id="s0080"><title>VLP-assay</title><p id="p0145">BHK-21 cells grown in 10 cm-diameter dishes were co-transfected with plasmids (pCG1) encoding the TGEV M, E and S proteins. At 24 h p.t. VLPs secreted into the culture medium were purified through a 25% sucrose cushion by ultracentrifugation at 35,000 rpm in an SW 41 rotor (Beckman Coulter) for 1 h at 4 °C. VLP pellets were solubilized in 50 µl 2×SDS sample puffer and subjected to SDS-PAGE. Cells were lysed in NP-40 lysis puffer and were also subjected to SDS-PAGE. SDS gels were transferred by a semi-dry technique (<xref rid="bib22" ref-type="bibr">Kyhse-Andersen, 1984</xref>) to nitrocellulose membranes (GE Healthcare) that were subsequently blocked overnight with 1% blocking reagent (Roche) in blocking buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5). S protein was detected with mAB 6A.C3 (1:200) and M protein with mAB 9D.B4 (1:200) followed by incubation with an anti-mouse peroxidase-conjugated antibody (Dako; 1:1000). The nitrocellulose membrane was incubated with a chemiluminescent peroxidase substrate (Thermo Scientific), and chemiluminescence was measured with a Chemi Doc system (Biorad).</p></sec><sec id="s0085"><title>Cell lysate fractionation on sucrose density gradients</title><p id="p0150">BHK-21 cells were seeded in 10 cm-diameter dishes and were transfected with pCG1-TGEV-Swt, pCG1-TGEV-S_C<sub>1–10</sub>A or empty vector (pCG1) one day later. At 24 h p.t. the cells were metabolically labeled with 100 µCi/ml [<sup>35</sup>S]-Met/Cys in methionine- and cysteine-free Dulbecco׳s modified Eagle׳s medium (Gibco) supplemented with 4 mM <sc>l</sc>-glutamine (PAA Laboratories) for 6 h. The cells were solubilized in 250 µl 6 mM dodecyl-β-<italic>m</italic>-maltoside (Sigma), 10 mM Tris, 150 mM NaCl, pH 7.5, and protease inhibitors (Complete; Roche) per dish. The cell lysates were homogenized by a needle (G21×1, 5″) and lysed for 3 h at 4 °C. One milliliter of the cell lysate was loaded onto a sucrose gradient that consisted of a 50%-sucrose-cushion at the bottom of the tube overlaid with a gradient of 10–30% sucrose (in 10 mM Tris, 150 mM NaCl, pH 7.5, 6 mM dodecyl-β-<italic>m</italic>-maltoside, and protease inhibitor). The gradient was centrifuged at 35,000 rpm for 18 h in an SW40 Ti rotor at 4 °C (Beckman Coulter) and then divided into 700 µl fractions. 700 µl NP-40 lysis buffer was added and S proteins were isolated by immunoprecipitation. 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(Ve 141/10.1) from the <funding-source id="gs1">German Research Foundation (DFG)</funding-source>. C.S.-W. is funded by the Emmy Noether Programme from the DFG.</p><p>This work was performed by S.G. in partial fulfillment of the requirements for the PhD degree from the University of Veterinary Medicine Hannover. We thank Sandra Bauer for technical assistance.</p><p>We thank L. Enjuanes for the monoclonal antibodies 6A.C3 against the TGEV S protein and 9D.B4 against the TGEV M protein. We thank R. Cattaneo for providing the pCG1 expression plasmid.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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