Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competen...
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| Veröffentlicht in: | Virus genes 2022-06, Vol.58 (3), p.172-179 |
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| creator | Lee, Se Yeong Kim, Do Woo Jung, Yong Tae |
| description | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with
gag-pol
and
env
genes. For
env
vector construction, SARS-CoV-2 SΔ19
env
was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19
env
-EGFP. When pCLXSN-
gag-pol
and pCLXSN-SΔ19
env
-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10
4
TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10
4
TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs. |
| doi_str_mv | 10.1007/s11262-022-01890-z |
| format | Article |
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gag-pol
and
env
genes. For
env
vector construction, SARS-CoV-2 SΔ19
env
was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19
env
-EGFP. When pCLXSN-
gag-pol
and pCLXSN-SΔ19
env
-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10
4
TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10
4
TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.</description><identifier>ISSN: 0920-8569</identifier><identifier>EISSN: 1572-994X</identifier><identifier>DOI: 10.1007/s11262-022-01890-z</identifier><identifier>PMID: 35322356</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Animals ; Antiviral agents ; Biomedical and Life Sciences ; Biomedicine ; Cell lines ; Chlorocebus aethiops ; Coronaviruses ; COVID-19 ; Gag protein ; Giant Cells ; HEK293 Cells ; Humans ; Lymphocytes T ; Medical Microbiology ; Original Paper ; Plant Sciences ; Producer cells ; SARS-CoV-2 - genetics ; Severe acute respiratory syndrome coronavirus 2 ; Spike Glycoprotein, Coronavirus - genetics ; Syncytia ; Transfection ; Vero Cells ; Virology ; Viruses</subject><ispartof>Virus genes, 2022-06, Vol.58 (3), p.172-179</ispartof><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022</rights><rights>2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.</rights><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c404t-d11937f3e5c6cd4bac676cb8185a0a7aff1e2d9aea03baca55b54a44c30c4133</citedby><cites>FETCH-LOGICAL-c404t-d11937f3e5c6cd4bac676cb8185a0a7aff1e2d9aea03baca55b54a44c30c4133</cites><orcidid>0000-0002-9014-9145</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s11262-022-01890-z$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11262-022-01890-z$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,777,781,882,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35322356$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lee, Se Yeong</creatorcontrib><creatorcontrib>Kim, Do Woo</creatorcontrib><creatorcontrib>Jung, Yong Tae</creatorcontrib><title>Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation</title><title>Virus genes</title><addtitle>Virus Genes</addtitle><addtitle>Virus Genes</addtitle><description>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with
gag-pol
and
env
genes. For
env
vector construction, SARS-CoV-2 SΔ19
env
was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19
env
-EGFP. When pCLXSN-
gag-pol
and pCLXSN-SΔ19
env
-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10
4
TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10
4
TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.</description><subject>Animals</subject><subject>Antiviral agents</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Cell lines</subject><subject>Chlorocebus aethiops</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>Gag protein</subject><subject>Giant Cells</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Lymphocytes T</subject><subject>Medical Microbiology</subject><subject>Original Paper</subject><subject>Plant Sciences</subject><subject>Producer cells</subject><subject>SARS-CoV-2 - genetics</subject><subject>Severe acute respiratory syndrome coronavirus 2</subject><subject>Spike Glycoprotein, Coronavirus - genetics</subject><subject>Syncytia</subject><subject>Transfection</subject><subject>Vero Cells</subject><subject>Virology</subject><subject>Viruses</subject><issn>0920-8569</issn><issn>1572-994X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kU9LHDEYh0OpdFfbL9CDDPTSSzR_Z5KLIItVQRBUpPQSspnMNutuMiaZhfXTm3WtVQ8eQg7v8z553_wA-I7RAUaoOUwYk5pARMrBQiL48AmMMW8IlJL9_gzGSBIEBa_lCOymNEcICUHYFzCinBJCeT0GfybBpxwHk13wVeiq6-OrazgJt5BUqXd3FvbJDm3I6962VbQ5hpWLelGtrMkhVs63g3F-VqW1N-vsdNWFuNQb21ew0-lFst-e7z1w8-vkZnIGLy5PzyfHF9AwxDJsMZa06ajlpjYtm2pTN7WZCiy4RrrRXYctaaW2GtFS1JxPOdOMGYoMw5TugaOtth-mS9sa63OZT_XRLXVcq6Cdelvx7q-ahZUSkhHMmiL4-SyI4X6wKaulS8YuFtrbMCRFakbE5hdFQX-8Q-dhiL5sV6hCNLIWGyHZUiaGlKLtXobBSG2SU9vkVElOPSWnHkrT_us1Xlr-RVUAugVSKfmZjf_f_kD7CPv0p1E</recordid><startdate>20220601</startdate><enddate>20220601</enddate><creator>Lee, Se Yeong</creator><creator>Kim, Do Woo</creator><creator>Jung, Yong Tae</creator><general>Springer US</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9014-9145</orcidid></search><sort><creationdate>20220601</creationdate><title>Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation</title><author>Lee, Se Yeong ; Kim, Do Woo ; Jung, Yong Tae</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c404t-d11937f3e5c6cd4bac676cb8185a0a7aff1e2d9aea03baca55b54a44c30c4133</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Animals</topic><topic>Antiviral agents</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Cell lines</topic><topic>Chlorocebus aethiops</topic><topic>Coronaviruses</topic><topic>COVID-19</topic><topic>Gag protein</topic><topic>Giant Cells</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Lymphocytes T</topic><topic>Medical Microbiology</topic><topic>Original Paper</topic><topic>Plant Sciences</topic><topic>Producer cells</topic><topic>SARS-CoV-2 - genetics</topic><topic>Severe acute respiratory syndrome coronavirus 2</topic><topic>Spike Glycoprotein, Coronavirus - genetics</topic><topic>Syncytia</topic><topic>Transfection</topic><topic>Vero Cells</topic><topic>Virology</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lee, Se Yeong</creatorcontrib><creatorcontrib>Kim, Do Woo</creatorcontrib><creatorcontrib>Jung, Yong Tae</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Virus genes</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lee, Se Yeong</au><au>Kim, Do Woo</au><au>Jung, Yong Tae</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation</atitle><jtitle>Virus genes</jtitle><stitle>Virus Genes</stitle><addtitle>Virus Genes</addtitle><date>2022-06-01</date><risdate>2022</risdate><volume>58</volume><issue>3</issue><spage>172</spage><epage>179</epage><pages>172-179</pages><issn>0920-8569</issn><eissn>1572-994X</eissn><abstract>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with
gag-pol
and
env
genes. For
env
vector construction, SARS-CoV-2 SΔ19
env
was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19
env
-EGFP. When pCLXSN-
gag-pol
and pCLXSN-SΔ19
env
-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 10
4
TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 10
4
TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>35322356</pmid><doi>10.1007/s11262-022-01890-z</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0002-9014-9145</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Virus genes, 2022-06, Vol.58 (3), p.172-179 |
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| subjects | Animals Antiviral agents Biomedical and Life Sciences Biomedicine Cell lines Chlorocebus aethiops Coronaviruses COVID-19 Gag protein Giant Cells HEK293 Cells Humans Lymphocytes T Medical Microbiology Original Paper Plant Sciences Producer cells SARS-CoV-2 - genetics Severe acute respiratory syndrome coronavirus 2 Spike Glycoprotein, Coronavirus - genetics Syncytia Transfection Vero Cells Virology Viruses |
| title | Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation |
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