Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M
Identifieur interne : 000472 ( PascalFrancis/Checkpoint ); précédent : 000471; suivant : 000473Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M
Auteurs : M. Oostra [Pays-Bas] ; C. A. M. De Haan [Pays-Bas] ; R. J. De Groot [Pays-Bas] ; P. J. M. Rottier [Pays-Bas]Source :
- Journal of virology [ 0022-538X ] ; 2006.
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- Pascal (Inist)
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Abstract
The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.
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De Haan</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></affiliation></author><author><name sortKey="De Groot, R J" sort="De Groot, R J" uniqKey="De Groot R" first="R. J." last="De Groot">R. J. De Groot</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></affiliation></author><author><name sortKey="Rottier, P J M" sort="Rottier, P J M" uniqKey="Rottier P" first="P. J. M." last="Rottier">P. J. M. 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Oostra</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></affiliation></author><author><name sortKey="De Haan, C A M" sort="De Haan, C A M" uniqKey="De Haan C" first="C. A. M." last="De Haan">C. A. M. De Haan</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></affiliation></author><author><name sortKey="De Groot, R J" sort="De Groot, R J" uniqKey="De Groot R" first="R. J." last="De Groot">R. J. De Groot</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></affiliation></author><author><name sortKey="Rottier, P J M" sort="Rottier, P J M" uniqKey="Rottier P" first="P. J. M." last="Rottier">P. J. M. Rottier</name><affiliation wicri:level="4"><inist:fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>Pays-Bas</country><placeName><settlement type="city">Utrecht</settlement><region nuts="2">Utrecht (province)</region></placeName><orgName type="university">Université d'Utrecht</orgName></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="2006">2006</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>Coronavirus</term><term>Membrane protein</term><term>Microbiology</term><term>Severe acute respiratory syndrome</term><term>Virology</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term><term>Protéine membranaire</term><term>Microbiologie</term><term>Virologie</term><term>Syndrome respiratoire aigu sévère</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.</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>80</s2></fA05><fA06><s2>5</s2></fA06><fA08 i1="01" i2="1" l="ENG"><s1>Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M</s1></fA08><fA11 i1="01" i2="1"><s1>OOSTRA (M.)</s1></fA11><fA11 i1="02" i2="1"><s1>DE HAAN (C. A. M.)</s1></fA11><fA11 i1="03" i2="1"><s1>DE GROOT (R. J.)</s1></fA11><fA11 i1="04" i2="1"><s1>ROTTIER (P. J. M.)</s1></fA11><fA14 i1="01"><s1>Virology Division, Department of Infectious Diseases and Immunology, Utrecht University</s1><s2>3584 CL Utrecht</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ></fA14><fA20><s1>2326-2336</s1></fA20><fA21><s1>2006</s1></fA21><fA23 i1="01"><s0>ENG</s0></fA23><fA43 i1="01"><s1>INIST</s1><s2>13592</s2><s5>354000115154500250</s5></fA43><fA44><s0>0000</s0><s1>© 2006 INIST-CNRS. All rights reserved.</s1></fA44><fA45><s0>66 ref.</s0></fA45><fA47 i1="01" i2="1"><s0>06-0141833</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>The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. 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Oostra</name></region><name sortKey="De Groot, R J" sort="De Groot, R J" uniqKey="De Groot R" first="R. J." last="De Groot">R. J. De Groot</name><name sortKey="De Haan, C A M" sort="De Haan, C A M" uniqKey="De Haan C" first="C. A. M." last="De Haan">C. A. M. De Haan</name><name sortKey="Rottier, P J M" sort="Rottier, P J M" uniqKey="Rottier P" first="P. J. M." last="Rottier">P. J. M. Rottier</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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