The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity
Identifieur interne : 000563 ( PascalFrancis/Corpus ); précédent : 000562; suivant : 000564The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity
Auteurs : Naina Barretto ; Dalia Jukneliene ; Kiira Ratia ; ZHONGBIN CHEN ; Andrew D. Mesecar ; Susan C. BakerSource :
- Journal of virology [ 0022-538X ] ; 2005.
Descripteurs français
- Pascal (Inist)
English descriptors
Abstract
Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.
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NO : | PASCAL 06-0040396 INIST |
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ET : | The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity |
AU : | BARRETTO (Naina); JUKNELIENE (Dalia); RATIA (Kiira); ZHONGBIN CHEN; MESECAR (Andrew D.); BAKER (Susan C.) |
AF : | Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine/Maywood, Illinois/Etats-Unis (1 aut., 2 aut., 4 aut., 6 aut.); Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago/Chicago, Illinois/Etats-Unis (3 aut., 5 aut.); Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine/Beijing/Chine (4 aut.) |
DT : | Publication en série; Niveau analytique |
SO : | Journal of virology; ISSN 0022-538X; Etats-Unis; Da. 2005; Vol. 79; No. 24; Pp. 15189-15198; Bibl. 63 ref. |
LA : | Anglais |
EA : | Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors. |
CC : | 002A05C10 |
FD : | Coronavirus; Papain; Microbiologie; Virologie; Syndrome respiratoire aigu sévère |
FG : | Coronaviridae; Nidovirales; Virus; Cysteine endopeptidases; Peptidases; Hydrolases; Enzyme; Appareil respiratoire pathologie; Virose; Infection; Poumon pathologie |
ED : | Coronavirus; Papain; Microbiology; Virology; Severe acute respiratory syndrome |
EG : | Coronaviridae; Nidovirales; Virus; Cysteine endopeptidases; Peptidases; Hydrolases; Enzyme; Respiratory disease; Viral disease; Infection; Lung disease |
SD : | Coronavirus; Papain; Microbiología; Virología; Síndrome respiratorio agudo severo |
LO : | INIST-13592.354000134506980200 |
ID : | 06-0040396 |
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<front><div type="abstract" xml:lang="en">Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.</div>
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<server><NO>PASCAL 06-0040396 INIST</NO>
<ET>The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity</ET>
<AU>BARRETTO (Naina); JUKNELIENE (Dalia); RATIA (Kiira); ZHONGBIN CHEN; MESECAR (Andrew D.); BAKER (Susan C.)</AU>
<AF>Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine/Maywood, Illinois/Etats-Unis (1 aut., 2 aut., 4 aut., 6 aut.); Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago/Chicago, Illinois/Etats-Unis (3 aut., 5 aut.); Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine/Beijing/Chine (4 aut.)</AF>
<DT>Publication en série; Niveau analytique</DT>
<SO>Journal of virology; ISSN 0022-538X; Etats-Unis; Da. 2005; Vol. 79; No. 24; Pp. 15189-15198; Bibl. 63 ref.</SO>
<LA>Anglais</LA>
<EA>Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.</EA>
<CC>002A05C10</CC>
<FD>Coronavirus; Papain; Microbiologie; Virologie; Syndrome respiratoire aigu sévère</FD>
<FG>Coronaviridae; Nidovirales; Virus; Cysteine endopeptidases; Peptidases; Hydrolases; Enzyme; Appareil respiratoire pathologie; Virose; Infection; Poumon pathologie</FG>
<ED>Coronavirus; Papain; Microbiology; Virology; Severe acute respiratory syndrome</ED>
<EG>Coronaviridae; Nidovirales; Virus; Cysteine endopeptidases; Peptidases; Hydrolases; Enzyme; Respiratory disease; Viral disease; Infection; Lung disease</EG>
<SD>Coronavirus; Papain; Microbiología; Virología; Síndrome respiratorio agudo severo</SD>
<LO>INIST-13592.354000134506980200</LO>
<ID>06-0040396</ID>
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