Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex.
Identifieur interne : 002223 ( PubMed/Curation ); précédent : 002222; suivant : 002224Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex.
Auteurs : Eric J. Snijder [Pays-Bas] ; Yvonne Van Der Meer ; Jessika Zevenhoven-Dobbe ; Jos J M. Onderwater ; Jannes Van Der Meulen ; Henk K. Koerten ; A Mieke MommaasSource :
- Journal of virology [ 0022-538X ] ; 2006.
Descripteurs français
- KwdFr :
- Animaux, Cellules Vero, Membranes intracellulaires (métabolisme), Membranes intracellulaires (ultrastructure), Microscopie électronique, Origine de réplication, RNA replicase (analyse), Réplication virale, Réticulum endoplasmique (microbiologie), Réticulum endoplasmique (virologie), Virus du SRAS, Vésicules de transport (ultrastructure).
- MESH :
- analyse : RNA replicase.
- microbiologie : Réticulum endoplasmique.
- métabolisme : Membranes intracellulaires.
- virologie : Réticulum endoplasmique.
- ultrastructure : Animaux, Cellules Vero, Membranes intracellulaires, Microscopie électronique, Origine de réplication, Réplication virale, Virus du SRAS, Vésicules de transport.
English descriptors
- KwdEn :
- Animals, Chlorocebus aethiops (physiology), Endoplasmic Reticulum (microbiology), Endoplasmic Reticulum (virology), Intracellular Membranes (metabolism), Intracellular Membranes (ultrastructure), Microscopy, Electron, RNA Replicase (analysis), Replication Origin, SARS Virus, Transport Vesicles (ultrastructure), Vero Cells, Virus Replication.
- MESH :
- chemical , analysis : RNA Replicase.
- metabolism : Intracellular Membranes.
- microbiology : Endoplasmic Reticulum.
- physiology : Chlorocebus aethiops.
- ultrastructure : Intracellular Membranes, Transport Vesicles.
- virology : Endoplasmic Reticulum.
- Animals, Microscopy, Electron, Replication Origin, SARS Virus, Vero Cells, Virus Replication.
Abstract
The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.
DOI: 10.1128/JVI.02501-05
PubMed: 16731931
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Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">16731931</PMID><DateCompleted><Year>2006</Year><Month>08</Month><Day>16</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0022-538X</ISSN><JournalIssue CitedMedium="Print"><Volume>80</Volume><Issue>12</Issue><PubDate><Year>2006</Year><Month>Jun</Month></PubDate></JournalIssue><Title>Journal of virology</Title><ISOAbbreviation>J. Virol.</ISOAbbreviation></Journal><ArticleTitle>Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex.</ArticleTitle><Pagination><MedlinePgn>5927-40</MedlinePgn></Pagination><Abstract><AbstractText>The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Snijder</LastName><ForeName>Eric J</ForeName><Initials>EJ</Initials><AffiliationInfo><Affiliation>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. e.j.snijder@lumc.nl</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>van der Meer</LastName><ForeName>Yvonne</ForeName><Initials>Y</Initials></Author><Author ValidYN="Y"><LastName>Zevenhoven-Dobbe</LastName><ForeName>Jessika</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Onderwater</LastName><ForeName>Jos J M</ForeName><Initials>JJ</Initials></Author><Author ValidYN="Y"><LastName>van der Meulen</LastName><ForeName>Jannes</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Koerten</LastName><ForeName>Henk K</ForeName><Initials>HK</Initials></Author><Author ValidYN="Y"><LastName>Mommaas</LastName><ForeName>A Mieke</ForeName><Initials>AM</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Virol</MedlineTA><NlmUniqueID>0113724</NlmUniqueID><ISSNLinking>0022-538X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>EC 2.7.7.48</RegistryNumber><NameOfSubstance UI="D012324">RNA 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