A novel cell culture system modeling the SARS-CoV-2 life cycle
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2)...
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| Veröffentlicht in: | PLoS pathogens 2021-03, Vol.17 (3), p.e1009439 |
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| creator | Ju, Xiaohui Zhu, Yunkai Wang, Yuyan Li, Jingrui Zhang, Jiaxing Gong, Mingli Ren, Wenlin Li, Sai Zhong, Jin Zhang, Linqi Zhang, Qiangfeng Cliff Zhang, Rong Ding, Qiang |
| description | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development. |
| doi_str_mv | 10.1371/journal.ppat.1009439 |
| format | Article |
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SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1009439</identifier><identifier>PMID: 33711082</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adenocarcinoma ; Antiviral Agents - pharmacology ; Artificial chromosomes ; Biology and life sciences ; Biotechnology ; Cell culture ; Cell Culture Techniques - methods ; Cloning ; Containment of Biohazards ; Coronaviruses ; COVID-19 ; COVID-19 - virology ; Deoxyribonucleic acid ; DNA ; DNA-directed RNA polymerase ; Experiments ; Flow cytometry ; Genetic aspects ; Genome, Viral - drug effects ; Genomes ; High-Throughput Screening Assays ; Humans ; Infections ; Laboratory tests ; Life cycles ; Life cycles (Biology) ; Medicine and health sciences ; Methods ; Microscopy ; N protein ; Nucleotides ; Packaging ; Physiological aspects ; Poly(A) ; Polyadenylation ; Polymerase chain reaction ; Proteins ; Reporter gene ; Research and Analysis Methods ; RNA polymerase ; SARS-CoV-2 - drug effects ; SARS-CoV-2 - genetics ; SARS-CoV-2 - growth & development ; SARS-CoV-2 - physiology ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Transcription ; Tumor cell lines ; Viral diseases ; Virus Replication - drug effects ; Virus research ; Viruses</subject><ispartof>PLoS pathogens, 2021-03, Vol.17 (3), p.e1009439</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Ju et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2021 Ju et al 2021 Ju et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c661t-44214e0205033a3189774062d39cf761bc75ed2c2db3f5964358d98aca76ad793</citedby><cites>FETCH-LOGICAL-c661t-44214e0205033a3189774062d39cf761bc75ed2c2db3f5964358d98aca76ad793</cites><orcidid>0000-0002-9353-0355 ; 0000-0002-4913-0338 ; 0000-0003-2941-4808 ; 0000-0002-7290-0935 ; 0000-0003-4931-509X ; 0000-0002-9552-0222 ; 0000-0002-3554-1275</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990224/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990224/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33711082$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ju, Xiaohui</creatorcontrib><creatorcontrib>Zhu, Yunkai</creatorcontrib><creatorcontrib>Wang, Yuyan</creatorcontrib><creatorcontrib>Li, Jingrui</creatorcontrib><creatorcontrib>Zhang, Jiaxing</creatorcontrib><creatorcontrib>Gong, Mingli</creatorcontrib><creatorcontrib>Ren, Wenlin</creatorcontrib><creatorcontrib>Li, Sai</creatorcontrib><creatorcontrib>Zhong, Jin</creatorcontrib><creatorcontrib>Zhang, Linqi</creatorcontrib><creatorcontrib>Zhang, Qiangfeng Cliff</creatorcontrib><creatorcontrib>Zhang, Rong</creatorcontrib><creatorcontrib>Ding, Qiang</creatorcontrib><title>A novel cell culture system modeling the SARS-CoV-2 life cycle</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.</description><subject>Adenocarcinoma</subject><subject>Antiviral Agents - pharmacology</subject><subject>Artificial chromosomes</subject><subject>Biology and life sciences</subject><subject>Biotechnology</subject><subject>Cell culture</subject><subject>Cell Culture Techniques - methods</subject><subject>Cloning</subject><subject>Containment of Biohazards</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>COVID-19 - virology</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA-directed RNA polymerase</subject><subject>Experiments</subject><subject>Flow cytometry</subject><subject>Genetic aspects</subject><subject>Genome, Viral - drug effects</subject><subject>Genomes</subject><subject>High-Throughput Screening Assays</subject><subject>Humans</subject><subject>Infections</subject><subject>Laboratory tests</subject><subject>Life cycles</subject><subject>Life cycles (Biology)</subject><subject>Medicine and health sciences</subject><subject>Methods</subject><subject>Microscopy</subject><subject>N protein</subject><subject>Nucleotides</subject><subject>Packaging</subject><subject>Physiological aspects</subject><subject>Poly(A)</subject><subject>Polyadenylation</subject><subject>Polymerase chain reaction</subject><subject>Proteins</subject><subject>Reporter gene</subject><subject>Research and Analysis Methods</subject><subject>RNA polymerase</subject><subject>SARS-CoV-2 - drug effects</subject><subject>SARS-CoV-2 - genetics</subject><subject>SARS-CoV-2 - growth & development</subject><subject>SARS-CoV-2 - physiology</subject><subject>Severe acute respiratory syndrome</subject><subject>Severe acute respiratory syndrome coronavirus 2</subject><subject>Transcription</subject><subject>Tumor cell lines</subject><subject>Viral diseases</subject><subject>Virus Replication - drug effects</subject><subject>Virus research</subject><subject>Viruses</subject><issn>1553-7374</issn><issn>1553-7366</issn><issn>1553-7374</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqVkttq3DAQhk1paQ7tG5TWkKtceKuzrJvAsvSwEFrItr0VsjR2vMjW1rJD9-2rdJ2QhfaiCCQx-v5fM8Nk2RuMFphK_H4bpqE3frHbmXGBEVKMqmfZKeacFpJK9vzJ_SQ7i3GLEMMUi5fZCU0GGJXkNLta5n24A59b8Gmb_DgNkMd9HKHLu-DAt32Tj7eQb5Y3m2IVfhQk920Nud1bD6-yF7XxEV7P53n2_eOHb6vPxfXXT-vV8rqwQuCxYIxgBoggjig1FJdKSoYEcVTZWgpcWcnBEUtcRWuuBKO8dKo01khhnFT0PHt38N35EPVcetSEYypKpRRJxPpAuGC2eje0nRn2OphW_wmEodFmGNuUs3Y1KVMvOFR1SoyDscw5Z8qKWABF6uR1Nf82VR04C_04GH9kevzSt7e6CXdaKoUIYcngYjYYws8J4viPlGeqMSmrtq9DMrNdG61eCl5KzDESiVr8hUrLQdfa0EPdpviR4PJIkJgRfo2NmWLU683Nf7Bfjll2YO0QYhygfmwIRvp-JB-K1PcjqeeRTLK3T5v5KHqYQfobHnXaPQ</recordid><startdate>20210301</startdate><enddate>20210301</enddate><creator>Ju, Xiaohui</creator><creator>Zhu, Yunkai</creator><creator>Wang, Yuyan</creator><creator>Li, Jingrui</creator><creator>Zhang, Jiaxing</creator><creator>Gong, Mingli</creator><creator>Ren, Wenlin</creator><creator>Li, Sai</creator><creator>Zhong, Jin</creator><creator>Zhang, Linqi</creator><creator>Zhang, Qiangfeng Cliff</creator><creator>Zhang, Rong</creator><creator>Ding, Qiang</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>ISN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>COVID</scope><scope>DWQXO</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>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-9353-0355</orcidid><orcidid>https://orcid.org/0000-0002-4913-0338</orcidid><orcidid>https://orcid.org/0000-0003-2941-4808</orcidid><orcidid>https://orcid.org/0000-0002-7290-0935</orcidid><orcidid>https://orcid.org/0000-0003-4931-509X</orcidid><orcidid>https://orcid.org/0000-0002-9552-0222</orcidid><orcidid>https://orcid.org/0000-0002-3554-1275</orcidid></search><sort><creationdate>20210301</creationdate><title>A novel cell culture system modeling the SARS-CoV-2 life cycle</title><author>Ju, Xiaohui ; Zhu, Yunkai ; Wang, Yuyan ; Li, Jingrui ; Zhang, Jiaxing ; Gong, Mingli ; Ren, Wenlin ; Li, Sai ; Zhong, Jin ; Zhang, Linqi ; Zhang, Qiangfeng Cliff ; Zhang, Rong ; Ding, Qiang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c661t-44214e0205033a3189774062d39cf761bc75ed2c2db3f5964358d98aca76ad793</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Adenocarcinoma</topic><topic>Antiviral Agents - 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SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33711082</pmid><doi>10.1371/journal.ppat.1009439</doi><orcidid>https://orcid.org/0000-0002-9353-0355</orcidid><orcidid>https://orcid.org/0000-0002-4913-0338</orcidid><orcidid>https://orcid.org/0000-0003-2941-4808</orcidid><orcidid>https://orcid.org/0000-0002-7290-0935</orcidid><orcidid>https://orcid.org/0000-0003-4931-509X</orcidid><orcidid>https://orcid.org/0000-0002-9552-0222</orcidid><orcidid>https://orcid.org/0000-0002-3554-1275</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | PLoS pathogens, 2021-03, Vol.17 (3), p.e1009439 |
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| recordid | cdi_plos_journals_2513689992 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; PubMed Central Open Access; Public Library of Science (PLoS) |
| subjects | Adenocarcinoma Antiviral Agents - pharmacology Artificial chromosomes Biology and life sciences Biotechnology Cell culture Cell Culture Techniques - methods Cloning Containment of Biohazards Coronaviruses COVID-19 COVID-19 - virology Deoxyribonucleic acid DNA DNA-directed RNA polymerase Experiments Flow cytometry Genetic aspects Genome, Viral - drug effects Genomes High-Throughput Screening Assays Humans Infections Laboratory tests Life cycles Life cycles (Biology) Medicine and health sciences Methods Microscopy N protein Nucleotides Packaging Physiological aspects Poly(A) Polyadenylation Polymerase chain reaction Proteins Reporter gene Research and Analysis Methods RNA polymerase SARS-CoV-2 - drug effects SARS-CoV-2 - genetics SARS-CoV-2 - growth & development SARS-CoV-2 - physiology Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Transcription Tumor cell lines Viral diseases Virus Replication - drug effects Virus research Viruses |
| title | A novel cell culture system modeling the SARS-CoV-2 life cycle |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-02T10%3A16%3A32IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20novel%20cell%20culture%20system%20modeling%20the%20SARS-CoV-2%20life%20cycle&rft.jtitle=PLoS%20pathogens&rft.au=Ju,%20Xiaohui&rft.date=2021-03-01&rft.volume=17&rft.issue=3&rft.spage=e1009439&rft.pages=e1009439-&rft.issn=1553-7374&rft.eissn=1553-7374&rft_id=info:doi/10.1371/journal.ppat.1009439&rft_dat=%3Cgale_plos_%3EA658715106%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2513689992&rft_id=info:pmid/33711082&rft_galeid=A658715106&rft_doaj_id=oai_doaj_org_article_df280045ebf4425eac4ddda8b2cee92f&rfr_iscdi=true |