Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein
Identifieur interne : 005F10 ( Main/Merge ); précédent : 005F09; suivant : 005F11Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein
Auteurs : HYUN CHUL SONG [Italie] ; Mi-Young Seo [Italie] ; Konrad Stadler [Italie] ; Byoung J. Yoo [Corée du Sud] ; Qui-Lim Choo [Italie] ; Stephen R. Coates [Italie] ; Yasushi Uematsu [Italie] ; Takashi Harada [Italie] ; Catherine E. Greer [Italie] ; John M. Polo [Italie] ; Piero Pileri [Italie] ; Markus Eickmann [Allemagne] ; Rino Rappuoli [Italie] ; Sergio Abrignani [Italie] ; Michael Houghton [Italie] ; Jang H. Han [Italie]Source :
- Journal of virology [ 0022-538X ] ; 2004.
Descripteurs français
- Pascal (Inist)
English descriptors
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Abstract
We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ∼500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en" level="a">Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein</title><author><name sortKey="Hyun Chul Song" sort="Hyun Chul Song" uniqKey="Hyun Chul Song" last="Hyun Chul Song">HYUN CHUL SONG</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Seo, Mi Young" sort="Seo, Mi Young" uniqKey="Seo M" first="Mi-Young" last="Seo">Mi-Young Seo</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Stadler, Konrad" sort="Stadler, Konrad" uniqKey="Stadler K" first="Konrad" last="Stadler">Konrad Stadler</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Yoo, Byoung J" sort="Yoo, Byoung J" uniqKey="Yoo B" first="Byoung J." last="Yoo">Byoung J. 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Greer</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Polo, John M" sort="Polo, John M" uniqKey="Polo J" first="John M." last="Polo">John M. 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Yoo</name><affiliation wicri:level="1"><inist:fA14 i1="03"><s1>Division of Natural Sciences, Daegu University</s1><s2>Kyungbuk</s2><s3>KOR</s3><sZ>4 aut.</sZ></inist:fA14><country>Corée du Sud</country><wicri:noRegion>Kyungbuk</wicri:noRegion></affiliation></author><author><name sortKey="Choo, Qui Lim" sort="Choo, Qui Lim" uniqKey="Choo Q" first="Qui-Lim" last="Choo">Qui-Lim Choo</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Coates, Stephen R" sort="Coates, Stephen R" uniqKey="Coates S" first="Stephen R." last="Coates">Stephen R. Coates</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Uematsu, Yasushi" sort="Uematsu, Yasushi" uniqKey="Uematsu Y" first="Yasushi" last="Uematsu">Yasushi Uematsu</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Harada, Takashi" sort="Harada, Takashi" uniqKey="Harada T" first="Takashi" last="Harada">Takashi Harada</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Greer, Catherine E" sort="Greer, Catherine E" uniqKey="Greer C" first="Catherine E." last="Greer">Catherine E. Greer</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Polo, John M" sort="Polo, John M" uniqKey="Polo J" first="John M." last="Polo">John M. Polo</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Pileri, Piero" sort="Pileri, Piero" uniqKey="Pileri P" first="Piero" last="Pileri">Piero Pileri</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Eickmann, Markus" sort="Eickmann, Markus" uniqKey="Eickmann M" first="Markus" last="Eickmann">Markus Eickmann</name><affiliation wicri:level="3"><inist:fA14 i1="04"><s1>Institute for Virology, University of Marburg</s1><s2>Marburg</s2><s3>DEU</s3><sZ>12 aut.</sZ></inist:fA14><country>Allemagne</country><placeName><region type="land" nuts="1">Hesse (Land)</region><region type="district" nuts="2">District de Giessen</region><settlement type="city">Marbourg</settlement></placeName></affiliation></author><author><name sortKey="Rappuoli, Rino" sort="Rappuoli, Rino" uniqKey="Rappuoli R" first="Rino" last="Rappuoli">Rino Rappuoli</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Abrignani, Sergio" sort="Abrignani, Sergio" uniqKey="Abrignani S" first="Sergio" last="Abrignani">Sergio Abrignani</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>IRIS, Chiron Vaccines</s1><s2>Siena</s2><s3>ITA</s3><sZ>3 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ><sZ>11 aut.</sZ><sZ>13 aut.</sZ><sZ>14 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Siena</wicri:noRegion></affiliation></author><author><name sortKey="Houghton, Michael" sort="Houghton, Michael" uniqKey="Houghton M" first="Michael" last="Houghton">Michael Houghton</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author><author><name sortKey="Han, Jang H" sort="Han, Jang H" uniqKey="Han J" first="Jang H." last="Han">Jang H. Han</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Vaccines Research, Chiron Corporation</s1><s2>Emeryville, California</s2><s3>ITA</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>9 aut.</sZ><sZ>10 aut.</sZ><sZ>13 aut.</sZ><sZ>15 aut.</sZ><sZ>16 aut.</sZ></inist:fA14><country>Italie</country><wicri:noRegion>Emeryville, California</wicri:noRegion></affiliation></author></analytic><series><title level="j" type="main">Journal of virology</title><title level="j" type="abbreviated">J. virol.</title><idno type="ISSN">0022-538X</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of virology</title><title level="j" type="abbreviated">J. virol.</title><idno type="ISSN">0022-538X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Acute</term><term>Characterization</term><term>Coronavirus</term><term>Critically ill</term><term>Glycoprotein</term><term>Microbiology</term><term>Oligomerization</term><term>Respiratory tract</term><term>Severe</term><term>Severe acute respiratory syndrome</term><term>Synthesis</term><term>Virology</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term><term>Synthèse</term><term>Caractérisation</term><term>Oligomérisation</term><term>Grave</term><term>Malade état grave</term><term>Aigu</term><term>Voie respiratoire</term><term>Glycoprotéine</term><term>Syndrome respiratoire aigu sévère</term><term>Microbiologie</term><term>Virologie</term><term>Forme grave</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ∼500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.</div></front></TEI><affiliations><list><country><li>Allemagne</li><li>Corée du Sud</li><li>Italie</li></country><region><li>District de Giessen</li><li>Hesse (Land)</li></region><settlement><li>Marbourg</li></settlement></list><tree><country name="Italie"><noRegion><name sortKey="Hyun Chul Song" sort="Hyun Chul Song" uniqKey="Hyun Chul Song" last="Hyun Chul Song">HYUN CHUL SONG</name></noRegion><name sortKey="Abrignani, Sergio" sort="Abrignani, Sergio" uniqKey="Abrignani S" first="Sergio" last="Abrignani">Sergio Abrignani</name><name sortKey="Choo, Qui Lim" sort="Choo, Qui Lim" uniqKey="Choo Q" first="Qui-Lim" last="Choo">Qui-Lim Choo</name><name sortKey="Coates, Stephen R" sort="Coates, Stephen R" uniqKey="Coates S" first="Stephen R." last="Coates">Stephen R. Coates</name><name sortKey="Greer, Catherine E" sort="Greer, Catherine E" uniqKey="Greer C" first="Catherine E." last="Greer">Catherine E. Greer</name><name sortKey="Han, Jang H" sort="Han, Jang H" uniqKey="Han J" first="Jang H." last="Han">Jang H. Han</name><name sortKey="Harada, Takashi" sort="Harada, Takashi" uniqKey="Harada T" first="Takashi" last="Harada">Takashi Harada</name><name sortKey="Houghton, Michael" sort="Houghton, Michael" uniqKey="Houghton M" first="Michael" last="Houghton">Michael Houghton</name><name sortKey="Pileri, Piero" sort="Pileri, Piero" uniqKey="Pileri P" first="Piero" last="Pileri">Piero Pileri</name><name sortKey="Polo, John M" sort="Polo, John M" uniqKey="Polo J" first="John M." last="Polo">John M. Polo</name><name sortKey="Rappuoli, Rino" sort="Rappuoli, Rino" uniqKey="Rappuoli R" first="Rino" last="Rappuoli">Rino Rappuoli</name><name sortKey="Rappuoli, Rino" sort="Rappuoli, Rino" uniqKey="Rappuoli R" first="Rino" last="Rappuoli">Rino Rappuoli</name><name sortKey="Seo, Mi Young" sort="Seo, Mi Young" uniqKey="Seo M" first="Mi-Young" last="Seo">Mi-Young Seo</name><name sortKey="Stadler, Konrad" sort="Stadler, Konrad" uniqKey="Stadler K" first="Konrad" last="Stadler">Konrad Stadler</name><name sortKey="Uematsu, Yasushi" sort="Uematsu, Yasushi" uniqKey="Uematsu Y" first="Yasushi" last="Uematsu">Yasushi Uematsu</name></country><country name="Corée du Sud"><noRegion><name sortKey="Yoo, Byoung J" sort="Yoo, Byoung J" uniqKey="Yoo B" first="Byoung J." last="Yoo">Byoung J. Yoo</name></noRegion></country><country name="Allemagne"><region name="Hesse (Land)"><name sortKey="Eickmann, Markus" sort="Eickmann, Markus" uniqKey="Eickmann M" first="Markus" last="Eickmann">Markus Eickmann</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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