A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.
Identifieur interne : 000658 ( PubMed/Corpus ); précédent : 000657; suivant : 000659A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.
Auteurs : Javier A. Jaimes ; Jean K. Millet ; Monty E. Goldstein ; Gary R. Whittaker ; Marco R. StrausSource :
- Journal of visualized experiments : JoVE [ 1940-087X ] ; 2019.
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
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><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></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></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><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></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><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; grw7@cornell.edu.</nlm:affiliation></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><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.</nlm:affiliation></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></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><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></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></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><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></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><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; grw7@cornell.edu.</nlm:affiliation></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><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.</nlm:affiliation></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="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" xml:lang="en"><term>Animals</term><term>Camelus</term><term>Humans</term><term>Proteolysis</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><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">30688313</PMID><DateCompleted><Year>2020</Year><Month>01</Month><Day>31</Day></DateCompleted><DateRevised><Year>2020</Year><Month>01</Month><Day>31</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1940-087X</ISSN><JournalIssue CitedMedium="Internet"><Issue>143</Issue><PubDate><Year>2019</Year><Month>01</Month><Day>09</Day></PubDate></JournalIssue><Title>Journal of visualized experiments : JoVE</Title><ISOAbbreviation>J Vis Exp</ISOAbbreviation></Journal><ArticleTitle>A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</ArticleTitle><ELocationID EIdType="doi" ValidYN="Y">10.3791/58892</ELocationID><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Jaimes</LastName><ForeName>Javier A</ForeName><Initials>JA</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Microbiology, College of Agricultural and Life Sciences, Cornell University.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Millet</LastName><ForeName>Jean K</ForeName><Initials>JK</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Virologie et Immunologie Moléculaires, Domaine de Vilvert, INRA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Goldstein</LastName><ForeName>Monty E</ForeName><Initials>ME</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; Department of Cell Biology and Molecular Genetics, University of Maryland.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Whittaker</LastName><ForeName>Gary R</ForeName><Initials>GR</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; grw7@cornell.edu.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Straus</LastName><ForeName>Marco R</ForeName><Initials>MR</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>R21 AI111085</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D052061">Research Support, N.I.H., Extramural</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D059040">Video-Audio Media</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>01</Month><Day>09</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Vis Exp</MedlineTA><NlmUniqueID>101313252</NlmUniqueID><ISSNLinking>1940-087X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010455">Peptides</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D064370">Spike Glycoprotein, Coronavirus</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002162" MajorTopicYN="N">Camelus</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D065207" MajorTopicYN="N">Middle East Respiratory Syndrome Coronavirus</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010455" MajorTopicYN="N">Peptides</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D059748" MajorTopicYN="Y">Proteolysis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D064370" MajorTopicYN="N">Spike Glycoprotein, Coronavirus</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>1</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>1</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>2</Month><Day>1</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30688313</ArticleId><ArticleId IdType="doi">10.3791/58892</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/PubMed/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000658 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd -nk 000658 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= PubMed
|étape= Corpus
|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/PubMed/Corpus/RBID.i -Sk "pubmed:30688313" \
| HfdSelect -Kh $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a MersV1
| This area was generated with Dilib version V0.6.33. |  |