SrasV1, PascalFrancis, Curation, bibRecord, 000427

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Identifieur interne : 000427 ( PascalFrancis/Curation ); précédent : 000426; suivant : 000428

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Auteurs : Naina Barretto [États-Unis] ; Dalia Jukneliene [États-Unis] ; Kiira Ratia [États-Unis] ; ZHONGBIN CHEN [États-Unis, République populaire de Chine] ; Andrew D. Mesecar [États-Unis] ; Susan C. Baker [États-Unis]

Source :

Journal of virology [ 0022-538X ] ; 2005.

RBID : Pascal:06-0040396

Descripteurs français

Pascal (Inist)
- Coronavirus, Papain, Microbiologie, Virologie, Syndrome respiratoire aigu sévère.

English descriptors

KwdEn :
- Coronavirus, Microbiology, Papain, Severe acute respiratory syndrome, Virology.

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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A11	`03`	`1`		`@1 RATIA (Kiira)`
A11	`04`	`1`		`@1 ZHONGBIN CHEN`
A11	`05`	`1`		`@1 MESECAR (Andrew D.)`
A11	`06`	`1`		`@1 BAKER (Susan C.)`
A14	`01`			`@1 Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine @2 Maywood, Illinois @3 USA @Z 1 aut. @Z 2 aut. @Z 4 aut. @Z 6 aut.`
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A14	`03`			`@1 Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine @2 Beijing @3 CHN @Z 4 aut.`
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C01	`01`		`ENG`	@0 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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Le document en format XML

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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity</title>
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<s2>Maywood, Illinois</s2>
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<author><name sortKey="Zhongbin Chen" sort="Zhongbin Chen" uniqKey="Zhongbin Chen" last="Zhongbin Chen">ZHONGBIN CHEN</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine</s1>
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<author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name>
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<seriesStmt><title level="j" type="main">Journal of virology</title>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Coronavirus</term>
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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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<fA11 i1="01" i2="1"><s1>BARRETTO (Naina)</s1>
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<fA11 i1="02" i2="1"><s1>JUKNELIENE (Dalia)</s1>
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<fA11 i1="03" i2="1"><s1>RATIA (Kiira)</s1>
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<fA11 i1="04" i2="1"><s1>ZHONGBIN CHEN</s1>
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<fA11 i1="05" i2="1"><s1>MESECAR (Andrew D.)</s1>
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<fA11 i1="06" i2="1"><s1>BAKER (Susan C.)</s1>
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<fA14 i1="01"><s1>Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine</s1>
<s2>Maywood, Illinois</s2>
<s3>USA</s3>
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<sZ>2 aut.</sZ>
<sZ>4 aut.</sZ>
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<fA14 i1="02"><s1>Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago</s1>
<s2>Chicago, Illinois</s2>
<s3>USA</s3>
<sZ>3 aut.</sZ>
<sZ>5 aut.</sZ>
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<fA14 i1="03"><s1>Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine</s1>
<s2>Beijing</s2>
<s3>CHN</s3>
<sZ>4 aut.</sZ>
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<fA66 i1="01"><s0>USA</s0>
</fA66>
<fC01 i1="01" l="ENG"><s0>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.</s0>
</fC01>
<fC02 i1="01" i2="X"><s0>002A05C10</s0>
</fC02>
<fC03 i1="01" i2="X" l="FRE"><s0>Coronavirus</s0>
<s2>NW</s2>
<s5>01</s5>
</fC03>
<fC03 i1="01" i2="X" l="ENG"><s0>Coronavirus</s0>
<s2>NW</s2>
<s5>01</s5>
</fC03>
<fC03 i1="01" i2="X" l="SPA"><s0>Coronavirus</s0>
<s2>NW</s2>
<s5>01</s5>
</fC03>
<fC03 i1="02" i2="X" l="FRE"><s0>Papain</s0>
<s2>FE</s2>
<s5>05</s5>
</fC03>
<fC03 i1="02" i2="X" l="ENG"><s0>Papain</s0>
<s2>FE</s2>
<s5>05</s5>
</fC03>
<fC03 i1="02" i2="X" l="SPA"><s0>Papain</s0>
<s2>FE</s2>
<s5>05</s5>
</fC03>
<fC03 i1="03" i2="X" l="FRE"><s0>Microbiologie</s0>
<s5>07</s5>
</fC03>
<fC03 i1="03" i2="X" l="ENG"><s0>Microbiology</s0>
<s5>07</s5>
</fC03>
<fC03 i1="03" i2="X" l="SPA"><s0>Microbiología</s0>
<s5>07</s5>
</fC03>
<fC03 i1="04" i2="X" l="FRE"><s0>Virologie</s0>
<s5>08</s5>
</fC03>
<fC03 i1="04" i2="X" l="ENG"><s0>Virology</s0>
<s5>08</s5>
</fC03>
<fC03 i1="04" i2="X" l="SPA"><s0>Virología</s0>
<s5>08</s5>
</fC03>
<fC03 i1="05" i2="X" l="FRE"><s0>Syndrome respiratoire aigu sévère</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC03 i1="05" i2="X" l="ENG"><s0>Severe acute respiratory syndrome</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC03 i1="05" i2="X" l="SPA"><s0>Síndrome respiratorio agudo severo</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC07 i1="01" i2="X" l="FRE"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="ENG"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="SPA"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="FRE"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="ENG"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="SPA"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="FRE"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="ENG"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="SPA"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="04" i2="X" l="FRE"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="04" i2="X" l="ENG"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="04" i2="X" l="SPA"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="FRE"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="ENG"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="SPA"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="FRE"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="ENG"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="SPA"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="FRE"><s0>Enzyme</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="ENG"><s0>Enzyme</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="SPA"><s0>Enzima</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="08" i2="X" l="FRE"><s0>Appareil respiratoire pathologie</s0>
<s5>13</s5>
</fC07>
<fC07 i1="08" i2="X" l="ENG"><s0>Respiratory disease</s0>
<s5>13</s5>
</fC07>
<fC07 i1="08" i2="X" l="SPA"><s0>Aparato respiratorio patología</s0>
<s5>13</s5>
</fC07>
<fC07 i1="09" i2="X" l="FRE"><s0>Virose</s0>
</fC07>
<fC07 i1="09" i2="X" l="ENG"><s0>Viral disease</s0>
</fC07>
<fC07 i1="09" i2="X" l="SPA"><s0>Virosis</s0>
</fC07>
<fC07 i1="10" i2="X" l="FRE"><s0>Infection</s0>
</fC07>
<fC07 i1="10" i2="X" l="ENG"><s0>Infection</s0>
</fC07>
<fC07 i1="10" i2="X" l="SPA"><s0>Infección</s0>
</fC07>
<fC07 i1="11" i2="X" l="FRE"><s0>Poumon pathologie</s0>
<s5>16</s5>
</fC07>
<fC07 i1="11" i2="X" l="ENG"><s0>Lung disease</s0>
<s5>16</s5>
</fC07>
<fC07 i1="11" i2="X" l="SPA"><s0>Pulmón patología</s0>
<s5>16</s5>
</fC07>
<fN21><s1>016</s1>
</fN21>
<fN44 i1="01"><s1>OTO</s1>
</fN44>
<fN82><s1>OTO</s1>
</fN82>
</pA>
</standard>
</inist>
</record>

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The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

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