Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins
Identifieur interne : 005B30 ( Main/Exploration ); précédent : 005B29; suivant : 005B31Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins
Auteurs : Erik Prentice [États-Unis] ; Josephine Mcauliffe [États-Unis] ; XIAOTAO LU [États-Unis] ; Kanta Subbarao [États-Unis] ; Mark R. Denison [États-Unis]Source :
- Journal of virology [ 0022-538X ] ; 2004.
Descripteurs français
- KwdFr :
- ADN viral (génétique), Animaux, Anticorps antiviraux, Cadres ouverts de lecture, Cellules Vero, Immunotransfert, Protéines virales (génétique), Protéines virales (immunologie), Protéines virales (isolement et purification), Protéines virales (métabolisme), RNA replicase (génétique), RNA replicase (immunologie), RNA replicase (isolement et purification), RNA replicase (métabolisme), Séquence nucléotidique, Technique d'immunofluorescence, Virus de l'hépatite murine (enzymologie), Virus de l'hépatite murine (génétique), Virus du SRAS (enzymologie), Virus du SRAS (génétique), Virus du SRAS (pathogénicité), Épitopes (génétique).
- MESH :
- enzymologie : Virus de l'hépatite murine, Virus du SRAS.
- génétique : ADN viral, Protéines virales, RNA replicase, Virus de l'hépatite murine, Virus du SRAS, Épitopes.
- immunologie : Protéines virales, RNA replicase.
- isolement et purification : Protéines virales, RNA replicase.
- métabolisme : Protéines virales, RNA replicase.
- pathogénicité : Virus du SRAS.
- Pascal (Inist)
- Animaux, Anticorps antiviraux, Cadres ouverts de lecture, Cellules Vero, Coronavirus, Identification, Caractérisation, Grave, Immunotransfert, Malade état grave, Aigu, Séquence nucléotidique, Technique d'immunofluorescence, Voie respiratoire, RNA-directed RNA polymerase, Protéine, Syndrome respiratoire aigu sévère, Microbiologie, Virologie, Forme grave.
English descriptors
- KwdEn :
- Acute, Animals, Antibodies, Viral, Base Sequence, Characterization, Chlorocebus aethiops, Coronavirus, Critically ill, DNA, Viral (genetics), Epitopes (genetics), Fluorescent Antibody Technique, Identification, Immunoblotting, Microbiology, Murine hepatitis virus (enzymology), Murine hepatitis virus (genetics), Open Reading Frames, Protein, RNA Replicase (genetics), RNA Replicase (immunology), RNA Replicase (isolation & purification), RNA Replicase (metabolism), RNA-directed RNA polymerase, Respiratory tract, SARS Virus (enzymology), SARS Virus (genetics), SARS Virus (pathogenicity), Severe, Severe acute respiratory syndrome, Vero Cells, Viral Proteins (genetics), Viral Proteins (immunology), Viral Proteins (isolation & purification), Viral Proteins (metabolism), Virology.
- MESH :
- chemical , genetics : DNA, Viral, Epitopes, RNA Replicase, Viral Proteins.
- chemical , immunology : RNA Replicase, Viral Proteins.
- chemical , isolation & purification : RNA Replicase, Viral Proteins.
- chemical , metabolism : RNA Replicase, Viral Proteins.
- chemical : Antibodies, Viral.
- enzymology : Murine hepatitis virus, SARS Virus.
- genetics : Murine hepatitis virus, SARS Virus.
- pathogenicity : SARS Virus.
- Animals, Base Sequence, Chlorocebus aethiops, Fluorescent Antibody Technique, Immunoblotting, Open Reading Frames, Vero Cells.
Abstract
The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nspl, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nspl4, nspl5, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nspl2 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.
Affiliations:
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Lamb Center for Pediatric Research, <sup>2</sup> Vanderbilt University Medical Center</s1><s2>Nashville, Tennessee</s2><s3>USA</s3><sZ>2 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Tennessee</region></placeName></affiliation></author><author><name sortKey="Xiaotao Lu" sort="Xiaotao Lu" uniqKey="Xiaotao Lu" last="Xiaotao Lu">XIAOTAO LU</name><affiliation wicri:level="2"><inist:fA14 i1="02"><s1>Department of Pediatrics, Vanderbilt University Medical Center</s1><s2>Nashville, Tennessee</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>3 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Tennessee</region></placeName></affiliation><affiliation wicri:level="2"><inist:fA14 i1="04"><s1>Laboratory of Infectious Diseases, National Institute of Allergy and Infections Diseases, National Institutes of Health</s1><s2>Bethesda, Maryland</s2><s3>USA</s3><sZ>3 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Maryland</region></placeName></affiliation></author><author><name sortKey="Subbarao, Kanta" sort="Subbarao, Kanta" uniqKey="Subbarao K" first="Kanta" last="Subbarao">Kanta Subbarao</name><affiliation wicri:level="2"><inist:fA14 i1="03"><s1>Elizabeth B. Lamb Center for Pediatric Research, <sup>2</sup> Vanderbilt University Medical Center</s1><s2>Nashville, Tennessee</s2><s3>USA</s3><sZ>2 aut.</sZ><sZ>4 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Tennessee</region></placeName></affiliation></author><author><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Department of Microbiology and Immunology, Vanderbilt University Medical Center</s1><s2>Nashville, Tennessee</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Tennessee</region></placeName></affiliation><affiliation wicri:level="2"><inist:fA14 i1="02"><s1>Department of Pediatrics, Vanderbilt University Medical Center</s1><s2>Nashville, Tennessee</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>3 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Tennessee</region></placeName></affiliation><affiliation wicri:level="2"><inist:fA14 i1="04"><s1>Laboratory of Infectious Diseases, National Institute of Allergy and Infections Diseases, National Institutes of Health</s1><s2>Bethesda, Maryland</s2><s3>USA</s3><sZ>3 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Maryland</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Journal of virology</title><title level="j" type="abbreviated">J. virol.</title><idno type="ISSN">0022-538X</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of virology</title><title level="j" type="abbreviated">J. virol.</title><idno type="ISSN">0022-538X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Acute</term><term>Animals</term><term>Antibodies, Viral</term><term>Base Sequence</term><term>Characterization</term><term>Chlorocebus aethiops</term><term>Coronavirus</term><term>Critically ill</term><term>DNA, Viral (genetics)</term><term>Epitopes (genetics)</term><term>Fluorescent Antibody Technique</term><term>Identification</term><term>Immunoblotting</term><term>Microbiology</term><term>Murine hepatitis virus (enzymology)</term><term>Murine hepatitis virus (genetics)</term><term>Open Reading Frames</term><term>Protein</term><term>RNA Replicase (genetics)</term><term>RNA Replicase (immunology)</term><term>RNA Replicase (isolation & purification)</term><term>RNA Replicase (metabolism)</term><term>RNA-directed RNA polymerase</term><term>Respiratory tract</term><term>SARS Virus (enzymology)</term><term>SARS Virus (genetics)</term><term>SARS Virus (pathogenicity)</term><term>Severe</term><term>Severe acute respiratory syndrome</term><term>Vero Cells</term><term>Viral Proteins (genetics)</term><term>Viral Proteins (immunology)</term><term>Viral Proteins (isolation & purification)</term><term>Viral Proteins (metabolism)</term><term>Virology</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN viral (génétique)</term><term>Animaux</term><term>Anticorps antiviraux</term><term>Cadres ouverts de lecture</term><term>Cellules Vero</term><term>Immunotransfert</term><term>Protéines virales (génétique)</term><term>Protéines virales (immunologie)</term><term>Protéines virales (isolement et purification)</term><term>Protéines virales (métabolisme)</term><term>RNA replicase (génétique)</term><term>RNA replicase (immunologie)</term><term>RNA replicase (isolement et purification)</term><term>RNA replicase (métabolisme)</term><term>Séquence nucléotidique</term><term>Technique d'immunofluorescence</term><term>Virus de l'hépatite murine (enzymologie)</term><term>Virus de l'hépatite murine (génétique)</term><term>Virus du SRAS (enzymologie)</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (pathogénicité)</term><term>Épitopes (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA, Viral</term><term>Epitopes</term><term>RNA Replicase</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>RNA Replicase</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>RNA Replicase</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>RNA Replicase</term><term>Viral Proteins</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Antibodies, Viral</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus de l'hépatite murine</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Murine hepatitis virus</term><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Murine hepatitis virus</term><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN viral</term><term>Protéines virales</term><term>RNA replicase</term><term>Virus de l'hépatite murine</term><term>Virus du SRAS</term><term>Épitopes</term></keywords><keywords scheme="MESH" qualifier="immunologie" xml:lang="fr"><term>Protéines virales</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines virales</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Protéines virales</term><term>RNA replicase</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" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Chlorocebus aethiops</term><term>Fluorescent Antibody Technique</term><term>Immunoblotting</term><term>Open Reading Frames</term><term>Vero Cells</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Animaux</term><term>Anticorps antiviraux</term><term>Cadres ouverts de lecture</term><term>Cellules Vero</term><term>Coronavirus</term><term>Identification</term><term>Caractérisation</term><term>Grave</term><term>Immunotransfert</term><term>Malade état grave</term><term>Aigu</term><term>Séquence nucléotidique</term><term>Technique d'immunofluorescence</term><term>Voie respiratoire</term><term>RNA-directed RNA polymerase</term><term>Protéine</term><term>Syndrome respiratoire aigu sévère</term><term>Microbiologie</term><term>Virologie</term><term>Forme grave</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nspl, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nspl4, nspl5, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nspl2 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Maryland</li><li>Tennessee</li></region></list><tree><country name="États-Unis"><region name="Tennessee"><name sortKey="Prentice, Erik" sort="Prentice, Erik" uniqKey="Prentice E" first="Erik" last="Prentice">Erik Prentice</name></region><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name><name sortKey="Mcauliffe, Josephine" sort="Mcauliffe, Josephine" uniqKey="Mcauliffe J" first="Josephine" last="Mcauliffe">Josephine Mcauliffe</name><name sortKey="Prentice, Erik" sort="Prentice, Erik" uniqKey="Prentice E" first="Erik" last="Prentice">Erik Prentice</name><name sortKey="Subbarao, Kanta" sort="Subbarao, Kanta" uniqKey="Subbarao K" first="Kanta" last="Subbarao">Kanta Subbarao</name><name sortKey="Xiaotao Lu" sort="Xiaotao Lu" uniqKey="Xiaotao Lu" last="Xiaotao Lu">XIAOTAO LU</name><name sortKey="Xiaotao Lu" sort="Xiaotao Lu" uniqKey="Xiaotao Lu" last="Xiaotao Lu">XIAOTAO LU</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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