Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein
Identifieur interne : 000194 ( PascalFrancis/Curation ); précédent : 000193; suivant : 000195Synthesis 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.
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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></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></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></affiliation></author><author><name sortKey="Yoo, Byoung J" sort="Yoo, Byoung J" uniqKey="Yoo B" first="Byoung J." last="Yoo">Byoung J. 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></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></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></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></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></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></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></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></affiliation></author><author><name sortKey="Eickmann, Markus" sort="Eickmann, Markus" uniqKey="Eickmann M" first="Markus" last="Eickmann">Markus Eickmann</name><affiliation wicri:level="1"><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></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></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></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></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></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></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">INIST</idno><idno type="inist">04-0528575</idno><date when="2004">2004</date><idno type="stanalyst">PASCAL 04-0528575 INIST</idno><idno type="RBID">Pascal:04-0528575</idno><idno type="wicri:Area/PascalFrancis/Corpus">000796</idno><idno type="wicri:Area/PascalFrancis/Curation">000194</idno></publicationStmt><sourceDesc><biblStruct><analytic><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></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></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></affiliation></author><author><name sortKey="Yoo, Byoung J" sort="Yoo, Byoung J" uniqKey="Yoo B" first="Byoung J." last="Yoo">Byoung J. 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></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></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></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></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></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></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></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></affiliation></author><author><name sortKey="Eickmann, Markus" sort="Eickmann, Markus" uniqKey="Eickmann M" first="Markus" last="Eickmann">Markus Eickmann</name><affiliation wicri:level="1"><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></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></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></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></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></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></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><inist><standard h6="B"><pA><fA01 i1="01" i2="1"><s0>0022-538X</s0></fA01><fA03 i2="1"><s0>J. virol.</s0></fA03><fA05><s2>78</s2></fA05><fA06><s2>19</s2></fA06><fA08 i1="01" i2="1" l="ENG"><s1>Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein</s1></fA08><fA11 i1="01" i2="1"><s1>HYUN CHUL SONG</s1></fA11><fA11 i1="02" i2="1"><s1>SEO (Mi-Young)</s1></fA11><fA11 i1="03" i2="1"><s1>STADLER (Konrad)</s1></fA11><fA11 i1="04" i2="1"><s1>YOO (Byoung J.)</s1></fA11><fA11 i1="05" i2="1"><s1>CHOO (Qui-Lim)</s1></fA11><fA11 i1="06" i2="1"><s1>COATES (Stephen R.)</s1></fA11><fA11 i1="07" i2="1"><s1>UEMATSU (Yasushi)</s1></fA11><fA11 i1="08" i2="1"><s1>HARADA (Takashi)</s1></fA11><fA11 i1="09" i2="1"><s1>GREER (Catherine E.)</s1></fA11><fA11 i1="10" i2="1"><s1>POLO (John M.)</s1></fA11><fA11 i1="11" i2="1"><s1>PILERI (Piero)</s1></fA11><fA11 i1="12" i2="1"><s1>EICKMANN (Markus)</s1></fA11><fA11 i1="13" i2="1"><s1>RAPPUOLI (Rino)</s1></fA11><fA11 i1="14" i2="1"><s1>ABRIGNANI (Sergio)</s1></fA11><fA11 i1="15" i2="1"><s1>HOUGHTON (Michael)</s1></fA11><fA11 i1="16" i2="1"><s1>HAN (Jang H.)</s1></fA11><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></fA14><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></fA14><fA14 i1="03"><s1>Division of Natural Sciences, Daegu University</s1><s2>Kyungbuk</s2><s3>KOR</s3><sZ>4 aut.</sZ></fA14><fA14 i1="04"><s1>Institute for Virology, University of Marburg</s1><s2>Marburg</s2><s3>DEU</s3><sZ>12 aut.</sZ></fA14><fA20><s1>10328-10335</s1></fA20><fA21><s1>2004</s1></fA21><fA23 i1="01"><s0>ENG</s0></fA23><fA43 i1="01"><s1>INIST</s1><s2>13592</s2><s5>354000122246720140</s5></fA43><fA44><s0>0000</s0><s1>© 2004 INIST-CNRS. All rights reserved.</s1></fA44><fA45><s0>36 ref.</s0></fA45><fA47 i1="01" i2="1"><s0>04-0528575</s0></fA47><fA60><s1>P</s1></fA60><fA61><s0>A</s0></fA61><fA64 i1="01" i2="1"><s0>Journal of virology</s0></fA64><fA66 i1="01"><s0>USA</s0></fA66><fC01 i1="01" l="ENG"><s0>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.</s0></fC01><fC02 i1="01" i2="X"><s0>002A05C10</s0></fC02><fC03 i1="01" i2="X" l="FRE"><s0>Coronavirus</s0><s2>NW</s2><s5>01</s5></fC03><fC03 i1="01" i2="X" l="ENG"><s0>Coronavirus</s0><s2>NW</s2><s5>01</s5></fC03><fC03 i1="01" i2="X" l="SPA"><s0>Coronavirus</s0><s2>NW</s2><s5>01</s5></fC03><fC03 i1="02" i2="X" l="FRE"><s0>Synthèse</s0><s5>05</s5></fC03><fC03 i1="02" i2="X" l="ENG"><s0>Synthesis</s0><s5>05</s5></fC03><fC03 i1="02" i2="X" l="SPA"><s0>Síntesis</s0><s5>05</s5></fC03><fC03 i1="03" i2="X" l="FRE"><s0>Caractérisation</s0><s5>06</s5></fC03><fC03 i1="03" i2="X" l="ENG"><s0>Characterization</s0><s5>06</s5></fC03><fC03 i1="03" i2="X" l="SPA"><s0>Caracterización</s0><s5>06</s5></fC03><fC03 i1="04" i2="X" l="FRE"><s0>Oligomérisation</s0><s5>07</s5></fC03><fC03 i1="04" i2="X" l="ENG"><s0>Oligomerization</s0><s5>07</s5></fC03><fC03 i1="04" i2="X" l="SPA"><s0>Oligomerización</s0><s5>07</s5></fC03><fC03 i1="05" i2="X" l="FRE"><s0>Grave</s0><s5>08</s5></fC03><fC03 i1="05" i2="X" l="ENG"><s0>Severe</s0><s5>08</s5></fC03><fC03 i1="05" i2="X" l="SPA"><s0>Grave</s0><s5>08</s5></fC03><fC03 i1="06" i2="X" l="FRE"><s0>Malade état grave</s0><s5>09</s5></fC03><fC03 i1="06" i2="X" l="ENG"><s0>Critically ill</s0><s5>09</s5></fC03><fC03 i1="06" i2="X" l="SPA"><s0>Enfermo estado grave</s0><s5>09</s5></fC03><fC03 i1="07" i2="X" l="FRE"><s0>Aigu</s0><s5>10</s5></fC03><fC03 i1="07" i2="X" l="ENG"><s0>Acute</s0><s5>10</s5></fC03><fC03 i1="07" i2="X" l="SPA"><s0>Agudo</s0><s5>10</s5></fC03><fC03 i1="08" i2="X" l="FRE"><s0>Voie respiratoire</s0><s5>11</s5></fC03><fC03 i1="08" i2="X" l="ENG"><s0>Respiratory tract</s0><s5>11</s5></fC03><fC03 i1="08" i2="X" l="SPA"><s0>Vía respiratoria</s0><s5>11</s5></fC03><fC03 i1="09" i2="X" l="FRE"><s0>Glycoprotéine</s0><s5>12</s5></fC03><fC03 i1="09" i2="X" l="ENG"><s0>Glycoprotein</s0><s5>12</s5></fC03><fC03 i1="09" i2="X" l="SPA"><s0>Glicoproteína</s0><s5>12</s5></fC03><fC03 i1="10" i2="X" l="FRE"><s0>Syndrome respiratoire aigu sévère</s0><s2>NM</s2><s5>14</s5></fC03><fC03 i1="10" i2="X" l="ENG"><s0>Severe acute respiratory syndrome</s0><s2>NM</s2><s5>14</s5></fC03><fC03 i1="10" i2="X" l="SPA"><s0>Síndrome respiratorio agudo severo</s0><s2>NM</s2><s5>14</s5></fC03><fC03 i1="11" i2="X" l="FRE"><s0>Microbiologie</s0><s5>67</s5></fC03><fC03 i1="11" i2="X" l="ENG"><s0>Microbiology</s0><s5>67</s5></fC03><fC03 i1="11" i2="X" l="SPA"><s0>Microbiología</s0><s5>67</s5></fC03><fC03 i1="12" i2="X" l="FRE"><s0>Virologie</s0><s5>68</s5></fC03><fC03 i1="12" i2="X" l="ENG"><s0>Virology</s0><s5>68</s5></fC03><fC03 i1="12" i2="X" l="SPA"><s0>Virología</s0><s5>68</s5></fC03><fC03 i1="13" i2="X" l="FRE"><s0>Forme grave</s0><s4>INC</s4><s5>79</s5></fC03><fC07 i1="01" i2="X" l="FRE"><s0>Coronaviridae</s0><s2>NW</s2></fC07><fC07 i1="01" i2="X" l="ENG"><s0>Coronaviridae</s0><s2>NW</s2></fC07><fC07 i1="01" i2="X" l="SPA"><s0>Coronaviridae</s0><s2>NW</s2></fC07><fC07 i1="02" i2="X" l="FRE"><s0>Nidovirales</s0><s2>NW</s2></fC07><fC07 i1="02" i2="X" l="ENG"><s0>Nidovirales</s0><s2>NW</s2></fC07><fC07 i1="02" i2="X" l="SPA"><s0>Nidovirales</s0><s2>NW</s2></fC07><fC07 i1="03" i2="X" l="FRE"><s0>Virus</s0><s2>NW</s2></fC07><fC07 i1="03" i2="X" l="ENG"><s0>Virus</s0><s2>NW</s2></fC07><fC07 i1="03" i2="X" l="SPA"><s0>Virus</s0><s2>NW</s2></fC07><fC07 i1="04" i2="X" l="FRE"><s0>Virose</s0><s2>NM</s2></fC07><fC07 i1="04" i2="X" l="ENG"><s0>Viral disease</s0><s2>NM</s2></fC07><fC07 i1="04" i2="X" l="SPA"><s0>Virosis</s0><s2>NM</s2></fC07><fC07 i1="05" i2="X" l="FRE"><s0>Infection</s0><s2>NM</s2></fC07><fC07 i1="05" i2="X" l="ENG"><s0>Infection</s0><s2>NM</s2></fC07><fC07 i1="05" i2="X" l="SPA"><s0>Infección</s0><s2>NM</s2></fC07><fC07 i1="06" i2="X" l="FRE"><s0>Appareil respiratoire pathologie</s0><s5>19</s5></fC07><fC07 i1="06" i2="X" l="ENG"><s0>Respiratory disease</s0><s5>19</s5></fC07><fC07 i1="06" i2="X" l="SPA"><s0>Aparato respiratorio patología</s0><s5>19</s5></fC07><fC07 i1="07" i2="X" l="FRE"><s0>Poumon pathologie</s0><s5>20</s5></fC07><fC07 i1="07" i2="X" l="ENG"><s0>Lung disease</s0><s5>20</s5></fC07><fC07 i1="07" i2="X" l="SPA"><s0>Pulmón patología</s0><s5>20</s5></fC07><fC07 i1="08" i2="X" l="FRE"><s0>Appareil respiratoire</s0><s5>22</s5></fC07><fC07 i1="08" i2="X" l="ENG"><s0>Respiratory system</s0><s5>22</s5></fC07><fC07 i1="08" i2="X" l="SPA"><s0>Aparato respiratorio</s0><s5>22</s5></fC07><fN21><s1>299</s1></fN21><fN44 i1="01"><s1>OTO</s1></fN44><fN82><s1>OTO</s1></fN82></pA></standard></inist></record>Pour manipuler ce document sous Unix (Dilib)
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