Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent.
Identifieur interne : 005405 ( Main/Exploration ); précédent : 005404; suivant : 005406Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent.
Auteurs : Berend Jan Bosch [Pays-Bas] ; Cornelis A M. De Haan ; Peter J M. RottierSource :
- Journal of virology [ 0022-538X ] ; 2004.
Descripteurs français
- KwdFr :
- Animaux, Assemblage viral, Données de séquences moléculaires, Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (), Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (métabolisme), Lignée cellulaire, Protéines de fusion recombinantes (métabolisme), Protéines de l'enveloppe virale (), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (métabolisme), Protéines luminescentes (génétique), Protéines luminescentes (métabolisme), Protéines à fluorescence verte, Recombinaison génétique, Souris, Séquence nucléotidique, Virion (métabolisme), Virus de l'hépatite murine (génétique), Virus de l'hépatite murine (métabolisme).
- MESH :
- génétique : Glycoprotéines membranaires, Protéines de l'enveloppe virale, Protéines luminescentes, Virus de l'hépatite murine.
- métabolisme : Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines luminescentes, Virion, Virus de l'hépatite murine.
- Animaux, Assemblage viral, Données de séquences moléculaires, Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires, Lignée cellulaire, Protéines de l'enveloppe virale, Protéines à fluorescence verte, Recombinaison génétique, Souris, Séquence nucléotidique.
English descriptors
- KwdEn :
- Animals, Base Sequence, Cell Line, Green Fluorescent Proteins, Luminescent Proteins (genetics), Luminescent Proteins (metabolism), Membrane Glycoproteins (chemistry), Membrane Glycoproteins (genetics), Membrane Glycoproteins (metabolism), Mice, Molecular Sequence Data, Murine hepatitis virus (genetics), Murine hepatitis virus (metabolism), Recombinant Fusion Proteins (metabolism), Recombination, Genetic, Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (chemistry), Viral Envelope Proteins (genetics), Viral Envelope Proteins (metabolism), Virion (metabolism), Virus Assembly.
- MESH :
- chemical , chemistry : Membrane Glycoproteins, Viral Envelope Proteins.
- chemical , genetics : Luminescent Proteins, Membrane Glycoproteins, Viral Envelope Proteins.
- chemical , metabolism : Luminescent Proteins, Membrane Glycoproteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemical : Green Fluorescent Proteins, Spike Glycoprotein, Coronavirus.
- genetics : Murine hepatitis virus.
- metabolism : Murine hepatitis virus, Virion.
- Animals, Base Sequence, Cell Line, Mice, Molecular Sequence Data, Recombination, Genetic, Virus Assembly.
Abstract
Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.
DOI: 10.1128/JVI.78.14.7369-7378.2004
PubMed: 15220410
Affiliations:
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Le document en format XML
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De Haan</name></author><author><name sortKey="Rottier, Peter J M" sort="Rottier, Peter J M" uniqKey="Rottier P" first="Peter J M" last="Rottier">Peter J M. Rottier</name></author></analytic><series><title level="j">Journal of virology</title><idno type="ISSN">0022-538X</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cell Line</term><term>Green Fluorescent Proteins</term><term>Luminescent Proteins (genetics)</term><term>Luminescent Proteins (metabolism)</term><term>Membrane Glycoproteins (chemistry)</term><term>Membrane Glycoproteins (genetics)</term><term>Membrane Glycoproteins (metabolism)</term><term>Mice</term><term>Molecular Sequence Data</term><term>Murine hepatitis virus (genetics)</term><term>Murine hepatitis virus (metabolism)</term><term>Recombinant Fusion Proteins (metabolism)</term><term>Recombination, Genetic</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (chemistry)</term><term>Viral Envelope Proteins (genetics)</term><term>Viral Envelope Proteins (metabolism)</term><term>Virion (metabolism)</term><term>Virus Assembly</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Assemblage viral</term><term>Données de séquences moléculaires</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires ()</term><term>Glycoprotéines membranaires (génétique)</term><term>Glycoprotéines membranaires (métabolisme)</term><term>Lignée cellulaire</term><term>Protéines de fusion recombinantes (métabolisme)</term><term>Protéines de l'enveloppe virale ()</term><term>Protéines de l'enveloppe virale (génétique)</term><term>Protéines de l'enveloppe virale (métabolisme)</term><term>Protéines luminescentes (génétique)</term><term>Protéines luminescentes (métabolisme)</term><term>Protéines à fluorescence verte</term><term>Recombinaison génétique</term><term>Souris</term><term>Séquence nucléotidique</term><term>Virion (métabolisme)</term><term>Virus de l'hépatite murine (génétique)</term><term>Virus de l'hépatite murine (métabolisme)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Membrane Glycoproteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Luminescent Proteins</term><term>Membrane Glycoproteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Luminescent Proteins</term><term>Membrane Glycoproteins</term><term>Recombinant Fusion Proteins</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Green Fluorescent Proteins</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Murine hepatitis virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Glycoprotéines membranaires</term><term>Protéines de l'enveloppe virale</term><term>Protéines luminescentes</term><term>Virus de l'hépatite murine</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Murine hepatitis virus</term><term>Virion</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Glycoprotéines membranaires</term><term>Protéines de fusion recombinantes</term><term>Protéines de l'enveloppe virale</term><term>Protéines luminescentes</term><term>Virion</term><term>Virus de l'hépatite murine</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cell Line</term><term>Mice</term><term>Molecular Sequence Data</term><term>Recombination, Genetic</term><term>Virus Assembly</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Assemblage viral</term><term>Données de séquences moléculaires</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires</term><term>Lignée cellulaire</term><term>Protéines de l'enveloppe virale</term><term>Protéines à fluorescence verte</term><term>Recombinaison génétique</term><term>Souris</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Utrecht (province)</li></region><settlement><li>Utrecht</li></settlement></list><tree><noCountry><name sortKey="De Haan, Cornelis A M" sort="De Haan, Cornelis A M" uniqKey="De Haan C" first="Cornelis A M" last="De Haan">Cornelis A M. De Haan</name><name sortKey="Rottier, Peter J M" sort="Rottier, Peter J M" uniqKey="Rottier P" first="Peter J M" last="Rottier">Peter J M. Rottier</name></noCountry><country name="Pays-Bas"><region name="Utrecht (province)"><name sortKey="Bosch, Berend Jan" sort="Bosch, Berend Jan" uniqKey="Bosch B" first="Berend Jan" last="Bosch">Berend Jan Bosch</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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