Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication
Identifieur interne : 000D87 ( Main/Exploration ); précédent : 000D86; suivant : 000D88Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication
Auteurs : Diede Oudshoorn [Pays-Bas] ; Kevin Rijs [Pays-Bas] ; Ronald W. A. L. Limpens [Pays-Bas] ; Kevin Groen [Pays-Bas] ; Abraham J. Koster [Pays-Bas] ; Eric J. Snijder [Pays-Bas] ; Marjolein Kikkert [Pays-Bas] ; Montserrat Bárcena [Pays-Bas]Source :
- mBio [ 2150-7511 ] ; 2017.
Descripteurs français
- KwdFr :
- ARN viral (métabolisme), Biogenèse des organelles, Coronavirus du syndrome respiratoire du Moyen-Orient (génétique), Coronavirus du syndrome respiratoire du Moyen-Orient (métabolisme), Humains, Lignée cellulaire, Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), Protéolyse, Réplication virale (génétique), Réticulum endoplasmique (virologie), Tomographie en microscopie électronique, Vésicules cytoplasmiques (métabolisme), Vésicules cytoplasmiques (ultrastructure), Vésicules cytoplasmiques (virologie).
- MESH :
- génétique : Coronavirus du syndrome respiratoire du Moyen-Orient, Protéines virales non structurales, Réplication virale.
- métabolisme : ARN viral, Coronavirus du syndrome respiratoire du Moyen-Orient, Protéines virales non structurales, Vésicules cytoplasmiques.
- virologie : Réticulum endoplasmique, Vésicules cytoplasmiques.
- ultrastructure : Biogenèse des organelles, Humains, Lignée cellulaire, Protéolyse, Tomographie en microscopie électronique, Vésicules cytoplasmiques.
English descriptors
- KwdEn :
- Cell Line, Cytoplasmic Vesicles (metabolism), Cytoplasmic Vesicles (ultrastructure), Cytoplasmic Vesicles (virology), Electron Microscope Tomography, Endoplasmic Reticulum (virology), Humans, Middle East Respiratory Syndrome Coronavirus (genetics), Middle East Respiratory Syndrome Coronavirus (metabolism), Organelle Biogenesis, Proteolysis, RNA, Viral (metabolism), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism), Virus Replication (genetics).
- MESH :
- chemical , genetics : Viral Nonstructural Proteins.
- chemical , metabolism : RNA, Viral, Viral Nonstructural Proteins.
- genetics : Middle East Respiratory Syndrome Coronavirus, Virus Replication.
- metabolism : Cytoplasmic Vesicles, Middle East Respiratory Syndrome Coronavirus.
- ultrastructure : Cytoplasmic Vesicles.
- virology : Cytoplasmic Vesicles, Endoplasmic Reticulum.
- Cell Line, Electron Microscope Tomography, Humans, Organelle Biogenesis, Proteolysis.
Abstract
Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.
Url:
DOI: 10.1128/mBio.01658-17
PubMed: 29162711
PubMed Central: 5698553
Affiliations:
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Koster</name><affiliation wicri:level="3"><nlm:aff id="aff2"><addr-line>Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</addr-line></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. 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Department of Medical Microbiology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author><author><name sortKey="Barcena, Montserrat" sort="Barcena, Montserrat" uniqKey="Barcena M" first="Montserrat" last="Bárcena">Montserrat Bárcena</name><affiliation wicri:level="3"><nlm:aff id="aff2"><addr-line>Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</addr-line></nlm:aff><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden</wicri:regionArea><placeName><settlement type="city">Leyde</settlement><region nuts="2" type="province">Hollande-Méridionale</region></placeName></affiliation></author></analytic><series><title level="j">mBio</title><idno type="eISSN">2150-7511</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cell Line</term><term>Cytoplasmic Vesicles (metabolism)</term><term>Cytoplasmic Vesicles (ultrastructure)</term><term>Cytoplasmic Vesicles (virology)</term><term>Electron Microscope Tomography</term><term>Endoplasmic Reticulum (virology)</term><term>Humans</term><term>Middle East Respiratory Syndrome Coronavirus (genetics)</term><term>Middle East Respiratory Syndrome Coronavirus (metabolism)</term><term>Organelle Biogenesis</term><term>Proteolysis</term><term>RNA, Viral (metabolism)</term><term>Viral Nonstructural Proteins (genetics)</term><term>Viral Nonstructural Proteins (metabolism)</term><term>Virus Replication (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (métabolisme)</term><term>Biogenèse des organelles</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (génétique)</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (métabolisme)</term><term>Humains</term><term>Lignée cellulaire</term><term>Protéines virales non structurales (génétique)</term><term>Protéines virales non structurales (métabolisme)</term><term>Protéolyse</term><term>Réplication virale (génétique)</term><term>Réticulum endoplasmique (virologie)</term><term>Tomographie en microscopie électronique</term><term>Vésicules cytoplasmiques (métabolisme)</term><term>Vésicules cytoplasmiques (ultrastructure)</term><term>Vésicules cytoplasmiques (virologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>RNA, Viral</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term><term>Virus Replication</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Protéines virales non structurales</term><term>Réplication virale</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Cytoplasmic Vesicles</term><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ARN viral</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Protéines virales non structurales</term><term>Vésicules cytoplasmiques</term></keywords><keywords scheme="MESH" qualifier="ultrastructure" xml:lang="en"><term>Cytoplasmic Vesicles</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Réticulum endoplasmique</term><term>Vésicules cytoplasmiques</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Cytoplasmic Vesicles</term><term>Endoplasmic Reticulum</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cell Line</term><term>Electron Microscope Tomography</term><term>Humans</term><term>Organelle Biogenesis</term><term>Proteolysis</term></keywords><keywords scheme="MESH" qualifier="ultrastructure" xml:lang="fr"><term>Biogenèse des organelles</term><term>Humains</term><term>Lignée cellulaire</term><term>Protéolyse</term><term>Tomographie en microscopie électronique</term><term>Vésicules cytoplasmiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>ABSTRACT</title><p>Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author><author><name sortKey="Gunther, S" uniqKey="Gunther S">S Günther</name></author><author><name sortKey="Preiser, W" uniqKey="Preiser W">W Preiser</name></author><author><name sortKey="Van Der Werf, S" uniqKey="Van Der Werf S">S van der Werf</name></author><author><name sortKey="Brodt, Hr" uniqKey="Brodt H">HR Brodt</name></author><author><name sortKey="Becker, S" uniqKey="Becker S">S Becker</name></author><author><name sortKey="Rabenau, H" uniqKey="Rabenau H">H Rabenau</name></author><author><name sortKey="Panning, M" uniqKey="Panning M">M Panning</name></author><author><name sortKey="Kolesnikova, 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McIntosh</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li></settlement></list><tree><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="Oudshoorn, Diede" sort="Oudshoorn, Diede" uniqKey="Oudshoorn D" first="Diede" last="Oudshoorn">Diede Oudshoorn</name></region><name sortKey="Barcena, Montserrat" sort="Barcena, Montserrat" uniqKey="Barcena M" first="Montserrat" last="Bárcena">Montserrat Bárcena</name><name sortKey="Groen, Kevin" sort="Groen, Kevin" uniqKey="Groen K" first="Kevin" last="Groen">Kevin Groen</name><name sortKey="Kikkert, Marjolein" sort="Kikkert, Marjolein" uniqKey="Kikkert M" first="Marjolein" last="Kikkert">Marjolein Kikkert</name><name sortKey="Koster, Abraham J" sort="Koster, Abraham J" uniqKey="Koster A" first="Abraham J." last="Koster">Abraham J. Koster</name><name sortKey="Limpens, Ronald W A L" sort="Limpens, Ronald W A L" uniqKey="Limpens R" first="Ronald W. A. L." last="Limpens">Ronald W. A. L. Limpens</name><name sortKey="Rijs, Kevin" sort="Rijs, Kevin" uniqKey="Rijs K" first="Kevin" last="Rijs">Kevin Rijs</name><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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