Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
Identifieur interne : 005B27 ( Main/Exploration ); précédent : 005B26; suivant : 005B28Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
Auteurs : Brian H. Harcourt [États-Unis] ; Dalia Jukneliene [États-Unis] ; Amornrat Kanjanahaluethai [États-Unis, Thaïlande] ; John Bechill [États-Unis] ; Kari M. Severson [États-Unis] ; Catherine M. Smith [États-Unis] ; Paul A. Rota [États-Unis] ; Susan C. Baker [États-Unis]Source :
- Journal of virology [ 0022-538X ] ; 2004.
Descripteurs français
- KwdFr :
- Animaux, Données de séquences moléculaires, Humains, Lignée cellulaire, Maturation post-traductionnelle des protéines, Mutation, Papaïne (), Papaïne (génétique), Papaïne (métabolisme), Polyprotéines (métabolisme), Protéines virales non structurales (), Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), RNA replicase (), RNA replicase (génétique), RNA replicase (métabolisme), Réplication virale, Séquence d'acides aminés, Virus du SRAS (enzymologie), Virus du SRAS (génétique), Virus du SRAS (physiologie).
- MESH :
- enzymologie : Virus du SRAS.
- génétique : Papaïne, Protéines virales non structurales, RNA replicase, Virus du SRAS.
- métabolisme : Papaïne, Polyprotéines, Protéines virales non structurales, RNA replicase.
- physiologie : Virus du SRAS.
- Pascal (Inist)
- Animaux, Coronavirus, Données de séquences moléculaires, Humains, Identification, Grave, Lignée cellulaire, Malade état grave, Aigu, Maturation post-traductionnelle des protéines, Mutation, Papaïne, Protéines virales non structurales, RNA replicase, Réplication virale, Séquence d'acides aminés, Voie respiratoire, RNA-directed RNA polymerase, Caractérisation, Papain, Syndrome respiratoire aigu sévère, Microbiologie, Virologie, Forme grave.
English descriptors
- KwdEn :
- Acute, Amino Acid Sequence, Animals, Cell Line, Characterization, Coronavirus, Critically ill, Humans, Identification, Microbiology, Molecular Sequence Data, Mutation, Papain, Papain (chemistry), Papain (genetics), Papain (metabolism), Polyproteins (metabolism), Protein Processing, Post-Translational, RNA Replicase (chemistry), RNA Replicase (genetics), RNA Replicase (metabolism), RNA-directed RNA polymerase, Respiratory tract, SARS Virus (enzymology), SARS Virus (genetics), SARS Virus (physiology), Severe, Severe acute respiratory syndrome, Viral Nonstructural Proteins (chemistry), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism), Virology, Virus Replication.
- MESH :
- chemical , chemistry : Papain, RNA Replicase, Viral Nonstructural Proteins.
- chemical , genetics : Papain, RNA Replicase, Viral Nonstructural Proteins.
- chemical , metabolism : Papain, Polyproteins, RNA Replicase, Viral Nonstructural Proteins.
- enzymology : SARS Virus.
- genetics : SARS Virus.
- physiology : SARS Virus.
- Amino Acid Sequence, Animals, Cell Line, Humans, Molecular Sequence Data, Mutation, Protein Processing, Post-Translational, Virus Replication.
Abstract
Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.
Affiliations:
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Severson</name><affiliation wicri:level="4"><inist:fA14 i1="02"><s1>Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago</s1><s2>Maywood, Illinois</s2><s3>USA</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Illinois</region><settlement type="city">Chicago</settlement></placeName><orgName type="university">Université de Chicago</orgName></affiliation></author><author><name sortKey="Smith, Catherine M" sort="Smith, Catherine M" uniqKey="Smith C" first="Catherine M." last="Smith">Catherine M. Smith</name><affiliation wicri:level="4"><inist:fA14 i1="02"><s1>Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago</s1><s2>Maywood, Illinois</s2><s3>USA</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Illinois</region><settlement type="city">Chicago</settlement></placeName><orgName type="university">Université de Chicago</orgName></affiliation></author><author><name sortKey="Rota, Paul A" sort="Rota, Paul A" uniqKey="Rota P" first="Paul A." last="Rota">Paul A. Rota</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Centers for Disease Control and Prevention</s1><s2>Atlanta, Georgia</s2><s3>USA</s3><sZ>1 aut.</sZ><sZ>7 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName></affiliation></author><author><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</name><affiliation wicri:level="4"><inist:fA14 i1="02"><s1>Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago</s1><s2>Maywood, Illinois</s2><s3>USA</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>États-Unis</country><placeName><region type="state">Illinois</region><settlement type="city">Chicago</settlement></placeName><orgName type="university">Université de Chicago</orgName></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>Amino Acid Sequence</term><term>Animals</term><term>Cell Line</term><term>Characterization</term><term>Coronavirus</term><term>Critically ill</term><term>Humans</term><term>Identification</term><term>Microbiology</term><term>Molecular Sequence Data</term><term>Mutation</term><term>Papain</term><term>Papain (chemistry)</term><term>Papain (genetics)</term><term>Papain (metabolism)</term><term>Polyproteins (metabolism)</term><term>Protein Processing, Post-Translational</term><term>RNA Replicase (chemistry)</term><term>RNA Replicase (genetics)</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 (physiology)</term><term>Severe</term><term>Severe acute respiratory syndrome</term><term>Viral Nonstructural Proteins (chemistry)</term><term>Viral Nonstructural Proteins (genetics)</term><term>Viral Nonstructural Proteins (metabolism)</term><term>Virology</term><term>Virus Replication</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Données de séquences moléculaires</term><term>Humains</term><term>Lignée cellulaire</term><term>Maturation post-traductionnelle des protéines</term><term>Mutation</term><term>Papaïne ()</term><term>Papaïne (génétique)</term><term>Papaïne (métabolisme)</term><term>Polyprotéines (métabolisme)</term><term>Protéines virales non structurales ()</term><term>Protéines virales non structurales (génétique)</term><term>Protéines virales non structurales (métabolisme)</term><term>RNA replicase ()</term><term>RNA replicase (génétique)</term><term>RNA replicase (métabolisme)</term><term>Réplication virale</term><term>Séquence d'acides aminés</term><term>Virus du SRAS (enzymologie)</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (physiologie)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Papain</term><term>RNA Replicase</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Papain</term><term>RNA Replicase</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Papain</term><term>Polyproteins</term><term>RNA Replicase</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Papaïne</term><term>Protéines virales non structurales</term><term>RNA replicase</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Papaïne</term><term>Polyprotéines</term><term>Protéines virales non structurales</term><term>RNA replicase</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>Cell Line</term><term>Humans</term><term>Molecular Sequence Data</term><term>Mutation</term><term>Protein Processing, Post-Translational</term><term>Virus Replication</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Animaux</term><term>Coronavirus</term><term>Données de séquences moléculaires</term><term>Humains</term><term>Identification</term><term>Grave</term><term>Lignée cellulaire</term><term>Malade état grave</term><term>Aigu</term><term>Maturation post-traductionnelle des protéines</term><term>Mutation</term><term>Papaïne</term><term>Protéines virales non structurales</term><term>RNA replicase</term><term>Réplication virale</term><term>Séquence d'acides aminés</term><term>Voie respiratoire</term><term>RNA-directed RNA polymerase</term><term>Caractérisation</term><term>Papain</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">Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.</div></front></TEI><affiliations><list><country><li>Thaïlande</li><li>États-Unis</li></country><region><li>Géorgie (États-Unis)</li><li>Illinois</li></region><settlement><li>Chicago</li></settlement><orgName><li>Université de Chicago</li></orgName></list><tree><country name="États-Unis"><region name="Géorgie (États-Unis)"><name sortKey="Harcourt, Brian H" sort="Harcourt, Brian H" uniqKey="Harcourt B" first="Brian H." last="Harcourt">Brian H. Harcourt</name></region><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</name><name sortKey="Bechill, John" sort="Bechill, John" uniqKey="Bechill J" first="John" last="Bechill">John Bechill</name><name sortKey="Jukneliene, Dalia" sort="Jukneliene, Dalia" uniqKey="Jukneliene D" first="Dalia" last="Jukneliene">Dalia Jukneliene</name><name sortKey="Kanjanahaluethai, Amornrat" sort="Kanjanahaluethai, Amornrat" uniqKey="Kanjanahaluethai A" first="Amornrat" last="Kanjanahaluethai">Amornrat Kanjanahaluethai</name><name sortKey="Rota, Paul A" sort="Rota, Paul A" uniqKey="Rota P" first="Paul A." last="Rota">Paul A. Rota</name><name sortKey="Severson, Kari M" sort="Severson, Kari M" uniqKey="Severson K" first="Kari M." last="Severson">Kari M. Severson</name><name sortKey="Smith, Catherine M" sort="Smith, Catherine M" uniqKey="Smith C" first="Catherine M." last="Smith">Catherine M. Smith</name></country><country name="Thaïlande"><noRegion><name sortKey="Kanjanahaluethai, Amornrat" sort="Kanjanahaluethai, Amornrat" uniqKey="Kanjanahaluethai A" first="Amornrat" last="Kanjanahaluethai">Amornrat Kanjanahaluethai</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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