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SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro
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...
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| Published in: | PLoS pathogens 2008-05, Vol.4 (5), p.e1000054 |
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| creator | van Hemert, Martijn J van den Worm, Sjoerd H E Knoops, Kèvin Mommaas, A Mieke Gorbalenya, Alexander E Snijder, Eric J |
| description | 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_str_mv | 10.1371/journal.ppat.1000054 |
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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.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1000054</identifier><identifier>PMID: 18451981</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Biochemistry/Macromolecular Assemblies and Machines ; Cell Biology/Membranes and Sorting ; Cercopithecus aethiops ; Cytoplasm - metabolism ; Dactinomycin - pharmacology ; Enzymes ; Gene Expression Regulation, Viral ; Genome, Viral ; Host-Pathogen Interactions ; Infectious Diseases/Viral Infections ; Microbiology ; Molecular Biology ; Proteins ; Rabbits ; Ribonucleic acid ; RNA ; RNA Processing, Post-Transcriptional ; RNA, Messenger - metabolism ; RNA, Viral - biosynthesis ; SARS Virus - pathogenicity ; SARS Virus - physiology ; SARS Virus - ultrastructure ; Transcription, Genetic - drug effects ; Transcription, Genetic - genetics ; Vero Cells ; Viral Matrix Proteins - metabolism ; Virology/Viral and Gene Regulation ; Virus Replication - physiology</subject><ispartof>PLoS pathogens, 2008-05, Vol.4 (5), p.e1000054</ispartof><rights>van Hemert et al. 2008</rights><rights>2008 van Hemert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: van Hemert MJ, van den Worm SHE, Knoops K, Mommaas AM, Gorbalenya AE, et al. (2008) SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro. PLoS Pathog 4(5): e1000054. doi:10.1371/journal.ppat.1000054</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c562t-799a1b35a42a218b6f8518137c7bed620ad3edd78d3ee60c01fc47f091e27a9f3</citedby><cites>FETCH-LOGICAL-c562t-799a1b35a42a218b6f8518137c7bed620ad3edd78d3ee60c01fc47f091e27a9f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322833/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322833/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,733,786,790,891,27957,27958,53827,53829</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18451981$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Baric, Ralph S.</contributor><creatorcontrib>van Hemert, Martijn J</creatorcontrib><creatorcontrib>van den Worm, Sjoerd H E</creatorcontrib><creatorcontrib>Knoops, Kèvin</creatorcontrib><creatorcontrib>Mommaas, A Mieke</creatorcontrib><creatorcontrib>Gorbalenya, Alexander E</creatorcontrib><creatorcontrib>Snijder, Eric J</creatorcontrib><title>SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>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.</description><subject>Animals</subject><subject>Biochemistry/Macromolecular Assemblies and Machines</subject><subject>Cell Biology/Membranes and Sorting</subject><subject>Cercopithecus aethiops</subject><subject>Cytoplasm - metabolism</subject><subject>Dactinomycin - pharmacology</subject><subject>Enzymes</subject><subject>Gene Expression Regulation, Viral</subject><subject>Genome, Viral</subject><subject>Host-Pathogen Interactions</subject><subject>Infectious Diseases/Viral Infections</subject><subject>Microbiology</subject><subject>Molecular Biology</subject><subject>Proteins</subject><subject>Rabbits</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA Processing, Post-Transcriptional</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Viral - biosynthesis</subject><subject>SARS Virus - pathogenicity</subject><subject>SARS Virus - physiology</subject><subject>SARS Virus - ultrastructure</subject><subject>Transcription, Genetic - drug effects</subject><subject>Transcription, Genetic - genetics</subject><subject>Vero Cells</subject><subject>Viral Matrix Proteins - metabolism</subject><subject>Virology/Viral and Gene Regulation</subject><subject>Virus Replication - physiology</subject><issn>1553-7374</issn><issn>1553-7366</issn><issn>1553-7374</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>DOA</sourceid><recordid>eNpVUdtq3DAQNaUht-YPSqsf8EY3S_ZLIYS2CQQKSfssZGmUaLEtI2mX5O8rd902EYg5o5lzBs2pqo8EbwiT5HIbdnHSw2aedd4QXE7D31WnpGlYLZnk71_hk-ospS3GnDAijqsT0vKGdC05rZ4fru4fahNimPTex11CEebBG519mC5z1FMy0c9LhkwY5wGeISEdAY0w9qUM9RxDBpPBIj1ZNMEC0FNIGTltcojIlVuQ3_v8gvyESozhQ3Xk9JDgYo3n1a9vX39e39R3P77fXl_d1aYRNNey6zTpWaM51ZS0vXBtQ9qyACN7sIJibRlYK9sSQGCDiTNcOtwRoFJ3jp1Xnw-68xCSWpeWFKFth5kQnSwdt4cOG_RWzdGPOr6ooL368xDio9IxezOAciCFhbax0jIuudBcENP3TlIJAsyi9WWdtutHsAamssLhjejbyuSf1GPYK8oobRkrAvwgYGJIKYL7xyVYLb7__YJafFer74X26fXc_6TVaPYbjC-wUg</recordid><startdate>20080502</startdate><enddate>20080502</enddate><creator>van Hemert, Martijn J</creator><creator>van den Worm, Sjoerd H E</creator><creator>Knoops, Kèvin</creator><creator>Mommaas, A Mieke</creator><creator>Gorbalenya, Alexander E</creator><creator>Snijder, Eric J</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20080502</creationdate><title>SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro</title><author>van Hemert, Martijn J ; van den Worm, Sjoerd H E ; Knoops, Kèvin ; Mommaas, A Mieke ; Gorbalenya, Alexander E ; Snijder, Eric J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c562t-799a1b35a42a218b6f8518137c7bed620ad3edd78d3ee60c01fc47f091e27a9f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Animals</topic><topic>Biochemistry/Macromolecular Assemblies and Machines</topic><topic>Cell Biology/Membranes and Sorting</topic><topic>Cercopithecus aethiops</topic><topic>Cytoplasm - metabolism</topic><topic>Dactinomycin - pharmacology</topic><topic>Enzymes</topic><topic>Gene Expression Regulation, Viral</topic><topic>Genome, Viral</topic><topic>Host-Pathogen Interactions</topic><topic>Infectious Diseases/Viral Infections</topic><topic>Microbiology</topic><topic>Molecular Biology</topic><topic>Proteins</topic><topic>Rabbits</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA Processing, Post-Transcriptional</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Viral - biosynthesis</topic><topic>SARS Virus - pathogenicity</topic><topic>SARS Virus - physiology</topic><topic>SARS Virus - ultrastructure</topic><topic>Transcription, Genetic - drug effects</topic><topic>Transcription, Genetic - genetics</topic><topic>Vero Cells</topic><topic>Viral Matrix Proteins - metabolism</topic><topic>Virology/Viral and Gene Regulation</topic><topic>Virus Replication - physiology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>van Hemert, Martijn J</creatorcontrib><creatorcontrib>van den Worm, Sjoerd H E</creatorcontrib><creatorcontrib>Knoops, Kèvin</creatorcontrib><creatorcontrib>Mommaas, A Mieke</creatorcontrib><creatorcontrib>Gorbalenya, Alexander E</creatorcontrib><creatorcontrib>Snijder, Eric J</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><collection>Open Access: DOAJ - Directory of Open Access Journals</collection><jtitle>PLoS pathogens</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>van Hemert, Martijn J</au><au>van den Worm, Sjoerd H E</au><au>Knoops, Kèvin</au><au>Mommaas, A Mieke</au><au>Gorbalenya, Alexander E</au><au>Snijder, Eric J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro</atitle><jtitle>PLoS pathogens</jtitle><addtitle>PLoS Pathog</addtitle><date>2008-05-02</date><risdate>2008</risdate><volume>4</volume><issue>5</issue><spage>e1000054</spage><pages>e1000054-</pages><issn>1553-7374</issn><issn>1553-7366</issn><eissn>1553-7374</eissn><notes>Conceived and designed the experiments: MV AG ES. Performed the experiments: MV SV KK ES. Analyzed the data: MV SV KK AM ES. Wrote the paper: MV AG ES.</notes><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>18451981</pmid><doi>10.1371/journal.ppat.1000054</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Biochemistry/Macromolecular Assemblies and Machines Cell Biology/Membranes and Sorting Cercopithecus aethiops Cytoplasm - metabolism Dactinomycin - pharmacology Enzymes Gene Expression Regulation, Viral Genome, Viral Host-Pathogen Interactions Infectious Diseases/Viral Infections Microbiology Molecular Biology Proteins Rabbits Ribonucleic acid RNA RNA Processing, Post-Transcriptional RNA, Messenger - metabolism RNA, Viral - biosynthesis SARS Virus - pathogenicity SARS Virus - physiology SARS Virus - ultrastructure Transcription, Genetic - drug effects Transcription, Genetic - genetics Vero Cells Viral Matrix Proteins - metabolism Virology/Viral and Gene Regulation Virus Replication - physiology |
| title | SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro |
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