Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting.
Identifieur interne : 002150 ( Ncbi/Merge ); précédent : 002149; suivant : 002151Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting.
Auteurs : Jean K. Millet ; Tiffany Tang [États-Unis] ; Lakshmi Nathan [États-Unis] ; Javier A. Jaimes [États-Unis] ; Hung-Lun Hsu ; Susan Daniel [États-Unis] ; Gary R. Whittaker [États-Unis]Source :
- Journal of visualized experiments : JoVE [ 1940-087X ] ; 2019.
Descripteurs français
- KwdFr :
- Animaux, Cellules HEK293, Confinement de risques biologiques, Coronavirus (pathogénicité), Coronavirus du syndrome respiratoire du Moyen-Orient (pathogénicité), Glycoprotéine de spicule des coronavirus (métabolisme), Humains, Pénétration virale (), Souris, Virion (pathogénicité), Virus du SRAS (pathogénicité).
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Spike Glycoprotein, Coronavirus.
- drug effects : Virus Internalization.
- pathogenicity : Coronavirus, Middle East Respiratory Syndrome Coronavirus, SARS Virus, Virion.
- Animals, Containment of Biohazards, HEK293 Cells, Humans, Mice.
Abstract
The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.
DOI: 10.3791/59010
PubMed: 30882796
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Millet</name><affiliation><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; INRA, Virologie et Immunologie Moléculaires.</nlm:affiliation><wicri:noCountry code="subField">Virologie et Immunologie Moléculaires</wicri:noCountry></affiliation></author><author><name sortKey="Tang, Tiffany" sort="Tang, Tiffany" uniqKey="Tang T" first="Tiffany" last="Tang">Tiffany Tang</name><affiliation wicri:level="4"><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, 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="Nathan, Lakshmi" sort="Nathan, Lakshmi" uniqKey="Nathan L" first="Lakshmi" last="Nathan">Lakshmi Nathan</name><affiliation wicri:level="4"><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, 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="Jaimes, Javier A" sort="Jaimes, Javier A" uniqKey="Jaimes J" first="Javier A" last="Jaimes">Javier A. 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Millet</name><affiliation><nlm:affiliation>Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University; INRA, Virologie et Immunologie Moléculaires.</nlm:affiliation><wicri:noCountry code="subField">Virologie et Immunologie Moléculaires</wicri:noCountry></affiliation></author><author><name sortKey="Tang, Tiffany" sort="Tang, Tiffany" uniqKey="Tang T" first="Tiffany" last="Tang">Tiffany Tang</name><affiliation wicri:level="4"><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, 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="Nathan, Lakshmi" sort="Nathan, Lakshmi" uniqKey="Nathan L" first="Lakshmi" last="Nathan">Lakshmi Nathan</name><affiliation wicri:level="4"><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, 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="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, 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="Hsu, Hung Lun" sort="Hsu, Hung Lun" uniqKey="Hsu H" first="Hung-Lun" last="Hsu">Hung-Lun Hsu</name><affiliation><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University; Horae Gene Therapy Center, University of Massachusetts Medical School.</nlm:affiliation><wicri:noCountry code="subField">University of Massachusetts Medical School</wicri:noCountry></affiliation></author><author><name sortKey="Daniel, Susan" sort="Daniel, Susan" uniqKey="Daniel S" first="Susan" last="Daniel">Susan Daniel</name><affiliation wicri:level="4"><nlm:affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, 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="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; gary.whittaker@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></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>Containment of Biohazards</term><term>Coronavirus (pathogenicity)</term><term>HEK293 Cells</term><term>Humans</term><term>Mice</term><term>Middle East Respiratory Syndrome Coronavirus (pathogenicity)</term><term>SARS Virus (pathogenicity)</term><term>Spike Glycoprotein, Coronavirus (metabolism)</term><term>Virion (pathogenicity)</term><term>Virus Internalization (drug effects)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Cellules HEK293</term><term>Confinement de risques biologiques</term><term>Coronavirus (pathogénicité)</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (pathogénicité)</term><term>Glycoprotéine de spicule des coronavirus (métabolisme)</term><term>Humains</term><term>Pénétration virale ()</term><term>Souris</term><term>Virion (pathogénicité)</term><term>Virus du SRAS (pathogénicité)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" qualifier="drug effects" xml:lang="en"><term>Virus Internalization</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Glycoprotéine de spicule des coronavirus</term></keywords><keywords scheme="MESH" qualifier="pathogenicity" xml:lang="en"><term>Coronavirus</term><term>Middle East Respiratory Syndrome Coronavirus</term><term>SARS Virus</term><term>Virion</term></keywords><keywords scheme="MESH" qualifier="pathogénicité" xml:lang="fr"><term>Coronavirus</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Virion</term><term>Virus du SRAS</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Containment of Biohazards</term><term>HEK293 Cells</term><term>Humans</term><term>Mice</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Cellules HEK293</term><term>Confinement de risques biologiques</term><term>Humains</term><term>Pénétration virale</term><term>Souris</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">30882796</PMID><DateCompleted><Year>2020</Year><Month>01</Month><Day>31</Day></DateCompleted><DateRevised><Year>2020</Year><Month>03</Month><Day>27</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1940-087X</ISSN><JournalIssue CitedMedium="Internet"><Issue>145</Issue><PubDate><Year>2019</Year><Month>03</Month><Day>01</Day></PubDate></JournalIssue><Title>Journal of visualized experiments : JoVE</Title><ISOAbbreviation>J Vis Exp</ISOAbbreviation></Journal><ArticleTitle>Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting.</ArticleTitle><ELocationID EIdType="doi" ValidYN="Y">10.3791/59010</ELocationID><Abstract><AbstractText>The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.</AbstractText></Abstract><AuthorList CompleteYN="Y"><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; INRA, Virologie et Immunologie Moléculaires.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Tang</LastName><ForeName>Tiffany</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Nathan</LastName><ForeName>Lakshmi</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Jaimes</LastName><ForeName>Javier A</ForeName><Initials>JA</Initials><AffiliationInfo><Affiliation>Department of Microbiology, College of Agricultural and Life Sciences, Cornell University.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hsu</LastName><ForeName>Hung-Lun</ForeName><Initials>HL</Initials><AffiliationInfo><Affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University; Horae Gene Therapy Center, University of Massachusetts Medical School.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Daniel</LastName><ForeName>Susan</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University.</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; gary.whittaker@cornell.edu.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>R01 AI135270</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant><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="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType><PublicationType UI="D059040">Video-Audio Media</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>03</Month><Day>01</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="D064370">Spike Glycoprotein, Coronavirus</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D003264" MajorTopicYN="Y">Containment of Biohazards</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017934" MajorTopicYN="N">Coronavirus</DescriptorName><QualifierName UI="Q000472" MajorTopicYN="Y">pathogenicity</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D057809" MajorTopicYN="N">HEK293 Cells</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D051379" MajorTopicYN="N">Mice</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D065207" MajorTopicYN="N">Middle East Respiratory Syndrome Coronavirus</DescriptorName><QualifierName UI="Q000472" MajorTopicYN="N">pathogenicity</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName><QualifierName UI="Q000472" MajorTopicYN="N">pathogenicity</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D064370" MajorTopicYN="N">Spike Glycoprotein, Coronavirus</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014771" MajorTopicYN="N">Virion</DescriptorName><QualifierName UI="Q000472" MajorTopicYN="N">pathogenicity</QualifierName></MeshHeading><MeshHeading><DescriptorName 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Millet</name></noCountry><country name="États-Unis"><region name="État de New York"><name sortKey="Tang, Tiffany" sort="Tang, Tiffany" uniqKey="Tang T" first="Tiffany" last="Tang">Tiffany Tang</name></region><name sortKey="Daniel, Susan" sort="Daniel, Susan" uniqKey="Daniel S" first="Susan" last="Daniel">Susan Daniel</name><name sortKey="Jaimes, Javier A" sort="Jaimes, Javier A" uniqKey="Jaimes J" first="Javier A" last="Jaimes">Javier A. Jaimes</name><name sortKey="Nathan, Lakshmi" sort="Nathan, Lakshmi" uniqKey="Nathan L" first="Lakshmi" last="Nathan">Lakshmi Nathan</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)
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