SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.
Identifieur interne : 003030 ( Main/Exploration ); précédent : 003029; suivant : 003031SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.
Auteurs : Martijn J. Van Hemert [Pays-Bas] ; Sjoerd H E. Van Den Worm ; Kèvin Knoops ; A Mieke Mommaas ; Alexander E. Gorbalenya ; Eric J. SnijderSource :
- PLoS pathogens [ 1553-7374 ] ; 2008.
Descripteurs français
- KwdFr :
- ARN messager (métabolisme), ARN viral (biosynthèse), Animaux, Cellules Vero, Cytoplasme (métabolisme), Dactinomycine (pharmacologie), Génome viral, Interactions hôte-pathogène, Lapins, Maturation post-transcriptionnelle des ARN, Protéines de la matrice virale (métabolisme), Régulation de l'expression des gènes viraux, Réplication virale (physiologie), Transcription génétique (), Transcription génétique (génétique), Virus du SRAS (pathogénicité), Virus du SRAS (physiologie), Virus du SRAS (ultrastructure).
- MESH :
- biosynthèse : ARN viral.
- génétique : Transcription génétique.
- métabolisme : ARN messager, Cytoplasme, Protéines de la matrice virale.
- pathogénicité : Virus du SRAS.
- pharmacologie : Dactinomycine.
- physiologie : Réplication virale, Virus du SRAS.
- Animaux, Cellules Vero, Génome viral, Interactions hôte-pathogène, Lapins, Maturation post-transcriptionnelle des ARN, Régulation de l'expression des gènes viraux, Transcription génétique, Virus du SRAS.
English descriptors
- KwdEn :
- Animals, Chlorocebus aethiops, Cytoplasm (metabolism), Dactinomycin (pharmacology), Gene Expression Regulation, Viral, Genome, Viral, Host-Pathogen Interactions, RNA Processing, Post-Transcriptional, RNA, Messenger (metabolism), RNA, Viral (biosynthesis), Rabbits, SARS Virus (pathogenicity), SARS Virus (physiology), SARS Virus (ultrastructure), Transcription, Genetic (drug effects), Transcription, Genetic (genetics), Vero Cells, Viral Matrix Proteins (metabolism), Virus Replication (physiology).
- MESH :
- chemical , biosynthesis : RNA, Viral.
- chemical , metabolism : RNA, Messenger, Viral Matrix Proteins.
- chemical , pharmacology : Dactinomycin.
- drug effects : Transcription, Genetic.
- genetics : Transcription, Genetic.
- metabolism : Cytoplasm.
- pathogenicity : SARS Virus.
- physiology : SARS Virus, Virus Replication.
- ultrastructure : SARS Virus.
- Animals, Chlorocebus aethiops, Gene Expression Regulation, Viral, Genome, Viral, Host-Pathogen Interactions, RNA Processing, Post-Transcriptional, Rabbits, Vero Cells.
Abstract
SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.
DOI: 10.1371/journal.ppat.1000054
PubMed: 18451981
Affiliations:
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Le document en format XML
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Van Hemert</name><affiliation wicri:level="3"><nlm:affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.</nlm:affiliation><country xml:lang="fr">Pays-Bas</country><wicri:regionArea>Molecular Virology Laboratory, 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="Van Den Worm, Sjoerd H E" sort="Van Den Worm, Sjoerd H E" uniqKey="Van Den Worm S" first="Sjoerd H E" last="Van Den Worm">Sjoerd H E. Van Den Worm</name></author><author><name sortKey="Knoops, Kevin" sort="Knoops, Kevin" uniqKey="Knoops K" first="Kèvin" last="Knoops">Kèvin Knoops</name></author><author><name sortKey="Mommaas, A Mieke" sort="Mommaas, A Mieke" uniqKey="Mommaas A" first="A Mieke" last="Mommaas">A Mieke Mommaas</name></author><author><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E" last="Gorbalenya">Alexander E. Gorbalenya</name></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name></author></analytic><series><title level="j">PLoS pathogens</title><idno type="eISSN">1553-7374</idno><imprint><date when="2008" type="published">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>Cytoplasm (metabolism)</term><term>Dactinomycin (pharmacology)</term><term>Gene Expression Regulation, Viral</term><term>Genome, Viral</term><term>Host-Pathogen Interactions</term><term>RNA Processing, Post-Transcriptional</term><term>RNA, Messenger (metabolism)</term><term>RNA, Viral (biosynthesis)</term><term>Rabbits</term><term>SARS Virus (pathogenicity)</term><term>SARS Virus (physiology)</term><term>SARS Virus (ultrastructure)</term><term>Transcription, Genetic (drug effects)</term><term>Transcription, Genetic (genetics)</term><term>Vero Cells</term><term>Viral Matrix Proteins (metabolism)</term><term>Virus Replication (physiology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (métabolisme)</term><term>ARN viral (biosynthèse)</term><term>Animaux</term><term>Cellules Vero</term><term>Cytoplasme (métabolisme)</term><term>Dactinomycine (pharmacologie)</term><term>Génome viral</term><term>Interactions hôte-pathogène</term><term>Lapins</term><term>Maturation post-transcriptionnelle des ARN</term><term>Protéines de la matrice virale (métabolisme)</term><term>Régulation de l'expression des gènes viraux</term><term>Réplication virale (physiologie)</term><term>Transcription génétique ()</term><term>Transcription génétique (génétique)</term><term>Virus du SRAS (pathogénicité)</term><term>Virus du SRAS (physiologie)</term><term>Virus du SRAS (ultrastructure)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>RNA, Viral</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>RNA, Messenger</term><term>Viral Matrix Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Dactinomycin</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>ARN viral</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Transcription génétique</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Cytoplasm</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>ARN messager</term><term>Cytoplasme</term><term>Protéines de la matrice virale</term></keywords><keywords scheme="MESH" qualifier="pathogenicity" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="pathogénicité" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Dactinomycine</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Réplication virale</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>SARS Virus</term><term>Virus Replication</term></keywords><keywords scheme="MESH" qualifier="ultrastructure" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>Gene Expression Regulation, Viral</term><term>Genome, Viral</term><term>Host-Pathogen Interactions</term><term>RNA Processing, Post-Transcriptional</term><term>Rabbits</term><term>Vero Cells</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules Vero</term><term>Génome viral</term><term>Interactions hôte-pathogène</term><term>Lapins</term><term>Maturation post-transcriptionnelle des ARN</term><term>Régulation de l'expression des gènes viraux</term><term>Transcription génétique</term><term>Virus du SRAS</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li></country><region><li>Hollande-Méridionale</li></region><settlement><li>Leyde</li></settlement></list><tree><noCountry><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E" last="Gorbalenya">Alexander E. Gorbalenya</name><name sortKey="Knoops, Kevin" sort="Knoops, Kevin" uniqKey="Knoops K" first="Kèvin" last="Knoops">Kèvin Knoops</name><name sortKey="Mommaas, A Mieke" sort="Mommaas, A Mieke" uniqKey="Mommaas A" first="A Mieke" last="Mommaas">A Mieke Mommaas</name><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J" last="Snijder">Eric J. Snijder</name><name sortKey="Van Den Worm, Sjoerd H E" sort="Van Den Worm, Sjoerd H E" uniqKey="Van Den Worm S" first="Sjoerd H E" last="Van Den Worm">Sjoerd H E. Van Den Worm</name></noCountry><country name="Pays-Bas"><region name="Hollande-Méridionale"><name sortKey="Van Hemert, Martijn J" sort="Van Hemert, Martijn J" uniqKey="Van Hemert M" first="Martijn J" last="Van Hemert">Martijn J. Van Hemert</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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