Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors.
Identifieur interne : 002050 ( Main/Exploration ); précédent : 002049; suivant : 002051Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors.
Auteurs : Andy Kilianski [États-Unis] ; Anna M. Mielech ; Xufang Deng ; Susan C. BakerSource :
- Journal of virology [ 1098-5514 ] ; 2013.
Descripteurs français
- KwdFr :
- Antiviraux (isolement et purification), Antiviraux (métabolisme), Coronavirus (), Coronavirus (enzymologie), Cysteine endopeptidases (métabolisme), Gènes rapporteurs, Humains, Inhibiteurs de protéases (isolement et purification), Inhibiteurs de protéases (métabolisme), Lignée cellulaire, Luciferases (analyse), Luciferases (génétique), Protéines virales (antagonistes et inhibiteurs), Protéines virales (métabolisme), Techniques de biocapteur (), Évaluation préclinique de médicament ().
- MESH :
- analyse : Luciferases.
- antagonistes et inhibiteurs : Protéines virales.
- enzymologie : Coronavirus.
- génétique : Luciferases.
- isolement et purification : Antiviraux, Inhibiteurs de protéases.
- métabolisme : Antiviraux, Cysteine endopeptidases, Inhibiteurs de protéases, Protéines virales.
- Coronavirus, Gènes rapporteurs, Humains, Lignée cellulaire, Techniques de biocapteur, Évaluation préclinique de médicament.
English descriptors
- KwdEn :
- Antiviral Agents (isolation & purification), Antiviral Agents (metabolism), Biosensing Techniques (methods), Cell Line, Coronavirus (drug effects), Coronavirus (enzymology), Cysteine Endopeptidases (metabolism), Drug Evaluation, Preclinical (methods), Genes, Reporter, Humans, Luciferases (analysis), Luciferases (genetics), Protease Inhibitors (isolation & purification), Protease Inhibitors (metabolism), Viral Proteins (antagonists & inhibitors), Viral Proteins (metabolism).
- MESH :
- chemical , analysis : Luciferases.
- chemical , antagonists & inhibitors : Viral Proteins.
- chemical , genetics : Luciferases.
- chemical , isolation & purification : Antiviral Agents, Protease Inhibitors.
- chemical , metabolism : Antiviral Agents, Cysteine Endopeptidases, Protease Inhibitors, Viral Proteins.
- drug effects : Coronavirus.
- enzymology : Coronavirus.
- methods : Biosensing Techniques, Drug Evaluation, Preclinical.
- Cell Line, Genes, Reporter, Humans.
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.
DOI: 10.1128/JVI.02105-13
PubMed: 23986593
Affiliations:
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Le document en format XML
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To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors. </div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Illinois</li></region><settlement><li>Chicago</li></settlement><orgName><li>Université de Chicago</li></orgName></list><tree><noCountry><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C" last="Baker">Susan C. Baker</name><name sortKey="Deng, Xufang" sort="Deng, Xufang" uniqKey="Deng X" first="Xufang" last="Deng">Xufang Deng</name><name sortKey="Mielech, Anna M" sort="Mielech, Anna M" uniqKey="Mielech A" first="Anna M" last="Mielech">Anna M. Mielech</name></noCountry><country name="États-Unis"><region name="Illinois"><name sortKey="Kilianski, Andy" sort="Kilianski, Andy" uniqKey="Kilianski A" first="Andy" last="Kilianski">Andy Kilianski</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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