A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.
Identifieur interne : 002093 ( Ncbi/Checkpoint ); précédent : 002092; suivant : 002094A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.
Auteurs : Javier A. Jaimes [États-Unis] ; Jean K. Millet ; Monty E. Goldstein [États-Unis] ; Gary R. Whittaker [États-Unis] ; Marco R. Straus [États-Unis]Source :
- Journal of visualized experiments : JoVE [ 1940-087X ] ; 2019.
Descripteurs français
- KwdFr :
- MESH :
- génétique : Coronavirus du syndrome respiratoire du Moyen-Orient, Glycoprotéine de spicule des coronavirus.
- métabolisme : Peptides.
- Animaux, Chameaux, Humains, Protéolyse.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Spike Glycoprotein, Coronavirus.
- chemical , metabolism : Peptides.
- genetics : Middle East Respiratory Syndrome Coronavirus.
- Animals, Camelus, Humans, Proteolysis.
Abstract
Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.
DOI: 10.3791/58892
PubMed: 30688313
Affiliations:
- États-Unis
- Maryland, État de New York
- College Park (Maryland), Ithaca (New York)
- Université Cornell, Université du Maryland
Links toward previous steps (curation, corpus...)
- to stream PubMed, to step Corpus: 000658
- to stream PubMed, to step Curation: 000658
- to stream PubMed, to step Checkpoint: 000710
- to stream Ncbi, to step Merge: 002093
- to stream Ncbi, to step Curation: 002093
Links to Exploration step
pubmed:30688313Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</title><author><name sortKey="Jaimes, Javier A" sort="Jaimes, Javier A" uniqKey="Jaimes J" first="Javier A" last="Jaimes">Javier A. Jaimes</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Microbiology, College of Agricultural and Life Sciences, Cornell University.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">Ithaca (New York)</settlement><region type="state">État de New York</region></placeName><orgName type="university">Université Cornell</orgName></affiliation></author><author><name sortKey="Millet, Jean K" sort="Millet, Jean K" uniqKey="Millet J" first="Jean K" last="Millet">Jean K. Millet</name><affiliation><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA.</nlm:affiliation><wicri:noCountry code="subField">INRA</wicri:noCountry></affiliation></author><author><name sortKey="Goldstein, Monty E" sort="Goldstein, Monty E" uniqKey="Goldstein M" first="Monty E" last="Goldstein">Monty E. Goldstein</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Cell Biology and Molecular Genetics, University of Maryland.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName><orgName type="university">Université du Maryland</orgName></affiliation></author><author><name sortKey="Whittaker, Gary R" sort="Whittaker, Gary R" uniqKey="Whittaker G" first="Gary R" last="Whittaker">Gary R. Whittaker</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; grw7@cornell.edu.</nlm:affiliation><country wicri:rule="url">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology, College of Veterinary Medicine</wicri:regionArea><wicri:noRegion>College of Veterinary Medicine</wicri:noRegion></affiliation></author><author><name sortKey="Straus, Marco R" sort="Straus, Marco R" uniqKey="Straus M" first="Marco R" last="Straus">Marco R. Straus</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">Ithaca (New York)</settlement><region type="state">État de New York</region></placeName><orgName type="university">Université Cornell</orgName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2019">2019</date><idno type="RBID">pubmed:30688313</idno><idno type="pmid">30688313</idno><idno type="doi">10.3791/58892</idno><idno type="wicri:Area/PubMed/Corpus">000658</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000658</idno><idno type="wicri:Area/PubMed/Curation">000658</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">000658</idno><idno type="wicri:Area/PubMed/Checkpoint">000710</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">000710</idno><idno type="wicri:Area/Ncbi/Merge">002093</idno><idno type="wicri:Area/Ncbi/Curation">002093</idno><idno type="wicri:Area/Ncbi/Checkpoint">002093</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</title><author><name sortKey="Jaimes, Javier A" sort="Jaimes, Javier A" uniqKey="Jaimes J" first="Javier A" last="Jaimes">Javier A. Jaimes</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Microbiology, College of Agricultural and Life Sciences, Cornell University.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">Ithaca (New York)</settlement><region type="state">État de New York</region></placeName><orgName type="university">Université Cornell</orgName></affiliation></author><author><name sortKey="Millet, Jean K" sort="Millet, Jean K" uniqKey="Millet J" first="Jean K" last="Millet">Jean K. Millet</name><affiliation><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA.</nlm:affiliation><wicri:noCountry code="subField">INRA</wicri:noCountry></affiliation></author><author><name sortKey="Goldstein, Monty E" sort="Goldstein, Monty E" uniqKey="Goldstein M" first="Monty E" last="Goldstein">Monty E. Goldstein</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Cell Biology and Molecular Genetics, University of Maryland.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName><orgName type="university">Université du Maryland</orgName></affiliation></author><author><name sortKey="Whittaker, Gary R" sort="Whittaker, Gary R" uniqKey="Whittaker G" first="Gary R" last="Whittaker">Gary R. Whittaker</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; grw7@cornell.edu.</nlm:affiliation><country wicri:rule="url">États-Unis</country><wicri:regionArea>Department of Microbiology and Immunology, College of Veterinary Medicine</wicri:regionArea><wicri:noRegion>College of Veterinary Medicine</wicri:noRegion></affiliation></author><author><name sortKey="Straus, Marco R" sort="Straus, Marco R" uniqKey="Straus M" first="Marco R" last="Straus">Marco R. Straus</name><affiliation wicri:level="4"><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.</nlm:affiliation><country>États-Unis</country><placeName><settlement type="city">Ithaca (New York)</settlement><region type="state">État de New York</region></placeName><orgName type="university">Université Cornell</orgName></affiliation></author></analytic><series><title level="j">Journal of visualized experiments : JoVE</title><idno type="eISSN">1940-087X</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Camelus</term><term>Humans</term><term>Middle East Respiratory Syndrome Coronavirus (genetics)</term><term>Peptides (metabolism)</term><term>Proteolysis</term><term>Spike Glycoprotein, Coronavirus (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Chameaux</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (génétique)</term><term>Glycoprotéine de spicule des coronavirus (génétique)</term><term>Humains</term><term>Peptides (métabolisme)</term><term>Protéolyse</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peptides</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Glycoprotéine de spicule des coronavirus</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Peptides</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Camelus</term><term>Humans</term><term>Proteolysis</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Chameaux</term><term>Humains</term><term>Protéolyse</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Maryland</li><li>État de New York</li></region><settlement><li>College Park (Maryland)</li><li>Ithaca (New York)</li></settlement><orgName><li>Université Cornell</li><li>Université du Maryland</li></orgName></list><tree><noCountry><name sortKey="Millet, Jean K" sort="Millet, Jean K" uniqKey="Millet J" first="Jean K" last="Millet">Jean K. Millet</name></noCountry><country name="États-Unis"><region name="État de New York"><name sortKey="Jaimes, Javier A" sort="Jaimes, Javier A" uniqKey="Jaimes J" first="Javier A" last="Jaimes">Javier A. Jaimes</name></region><name sortKey="Goldstein, Monty E" sort="Goldstein, Monty E" uniqKey="Goldstein M" first="Monty E" last="Goldstein">Monty E. Goldstein</name><name sortKey="Straus, Marco R" sort="Straus, Marco R" uniqKey="Straus M" first="Marco R" last="Straus">Marco R. Straus</name><name sortKey="Whittaker, Gary R" sort="Whittaker, Gary R" uniqKey="Whittaker G" first="Gary R" last="Whittaker">Gary R. Whittaker</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Ncbi/Checkpoint
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002093 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Ncbi/Checkpoint/biblio.hfd -nk 002093 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Ncbi
|étape= Checkpoint
|type= RBID
|clé= pubmed:30688313
|texte= A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Ncbi/Checkpoint/RBID.i -Sk "pubmed:30688313" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Ncbi/Checkpoint/biblio.hfd \
| NlmPubMed2Wicri -a MersV1
| This area was generated with Dilib version V0.6.33. |  |