Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication
Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RN...
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| Veröffentlicht in: | PloS one 2015-07, Vol.10 (7), p.e0133558-e0133558 |
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| creator | Wang, Zhen Zhao, Fei Gao, Qian Liu, Zhenlong Zhang, Yongxin Li, Xiaoyu Li, Yuhuan Ma, Weilie Deng, Tao Zhang, Zhizhen Cen, Shan |
| description | Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication. |
| doi_str_mv | 10.1371/journal.pone.0133558 |
| format | Article |
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Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0133558</identifier><identifier>PMID: 26196128</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Antiviral agents ; Assaying ; Biochemistry ; Biotechnology ; Cell Line, Tumor ; Cell Survival ; DNA-directed RNA polymerase ; Drug development ; Drug resistance ; Drug Resistance, Viral - genetics ; Drugs ; Gaussia ; Gene expression ; Genes, Reporter ; Genetic research ; Genome ; Genome, Viral ; Genomes ; Health risks ; HEK293 Cells ; High-throughput screening ; Humans ; Immunization ; Influenza ; Influenza A ; Influenza A virus - genetics ; Influenza A virus - physiology ; Influenza, Human - virology ; Inhibitors ; Luciferase ; Luciferases - metabolism ; Plasmids ; Proteins ; Public health ; Pyrrolidinones ; Replication ; Reproducibility ; Reproducibility of Results ; Ribonucleic acid ; RNA ; RNA Replicase - genetics ; RNA viruses ; RNA, Small Interfering - metabolism ; RNA, Viral - genetics ; RNA-directed RNA polymerase ; Sensitivity analysis ; Statistical analysis ; Transcription ; Transcription (Genetics) ; Transcription, Genetic ; Viral infections ; Virology ; Virus replication ; Virus Replication - drug effects ; Viruses</subject><ispartof>PloS one, 2015-07, Vol.10 (7), p.e0133558-e0133558</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Wang 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>2015 Wang et al 2015 Wang et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-1c44038c02fb05503ef356f8171233f6ec1134dd5007b10baa87d602ec851b403</citedby><cites>FETCH-LOGICAL-c758t-1c44038c02fb05503ef356f8171233f6ec1134dd5007b10baa87d602ec851b403</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511416/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511416/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26196128$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Digard, Paul</contributor><creatorcontrib>Wang, Zhen</creatorcontrib><creatorcontrib>Zhao, Fei</creatorcontrib><creatorcontrib>Gao, Qian</creatorcontrib><creatorcontrib>Liu, Zhenlong</creatorcontrib><creatorcontrib>Zhang, Yongxin</creatorcontrib><creatorcontrib>Li, Xiaoyu</creatorcontrib><creatorcontrib>Li, Yuhuan</creatorcontrib><creatorcontrib>Ma, Weilie</creatorcontrib><creatorcontrib>Deng, Tao</creatorcontrib><creatorcontrib>Zhang, Zhizhen</creatorcontrib><creatorcontrib>Cen, Shan</creatorcontrib><title>Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.</description><subject>Analysis</subject><subject>Antiviral agents</subject><subject>Assaying</subject><subject>Biochemistry</subject><subject>Biotechnology</subject><subject>Cell Line, Tumor</subject><subject>Cell Survival</subject><subject>DNA-directed RNA polymerase</subject><subject>Drug development</subject><subject>Drug resistance</subject><subject>Drug Resistance, Viral - genetics</subject><subject>Drugs</subject><subject>Gaussia</subject><subject>Gene expression</subject><subject>Genes, Reporter</subject><subject>Genetic research</subject><subject>Genome</subject><subject>Genome, Viral</subject><subject>Genomes</subject><subject>Health risks</subject><subject>HEK293 Cells</subject><subject>High-throughput screening</subject><subject>Humans</subject><subject>Immunization</subject><subject>Influenza</subject><subject>Influenza A</subject><subject>Influenza A virus - genetics</subject><subject>Influenza A virus - physiology</subject><subject>Influenza, Human - virology</subject><subject>Inhibitors</subject><subject>Luciferase</subject><subject>Luciferases - metabolism</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Public health</subject><subject>Pyrrolidinones</subject><subject>Replication</subject><subject>Reproducibility</subject><subject>Reproducibility of Results</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA Replicase - genetics</subject><subject>RNA viruses</subject><subject>RNA, Small Interfering - metabolism</subject><subject>RNA, Viral - genetics</subject><subject>RNA-directed RNA polymerase</subject><subject>Sensitivity analysis</subject><subject>Statistical analysis</subject><subject>Transcription</subject><subject>Transcription (Genetics)</subject><subject>Transcription, Genetic</subject><subject>Viral infections</subject><subject>Virology</subject><subject>Virus replication</subject><subject>Virus Replication - drug effects</subject><subject>Viruses</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</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><sourceid>DOA</sourceid><recordid>eNqNk11v0zAUhiMEYmPwDxBYQkJw0eKP2ElukKppsEqDSaXs1nIcO_HkxpntIMavx12zqUW7QL7w1_O-xz72ybLXCM4RKdCnazf6Xtj54Ho1h4gQSssn2TGqCJ4xDMnTvfFR9iKEawgpKRl7nh1hhiqGcHmctWchitqa0G1UH4HTQIBz03azdefd2HbDGMEiBHELogPfXG-i82DZazuq_o8AC3Bl_BjA6vsCrL3og_RmiMb1QPQNWKnBGim285fZMy1sUK-m_iT7-eVsfXo-u7j8ujxdXMxkQcs4QzLPISklxLqGlEKiNKFMl6hAmBDNlESI5E1DISxqBGshyqJhECtZUlQn6Un2duc7WBf4lKPAEauKglY4zxOx3BGNE9d88GYj_C13wvC7BedbLnw00iqeV5Lpmmlc4CqntErnqipdFQIXuhK0Tl6fp2hjvVGNTCn0wh6YHu70puOt-8VzilCOWDL4MBl4dzOqEPnGBKmsFb1y4925qzy9IiMJffcP-vjtJqoV6QKm1y7FlVtTvsgxpowSViZq_giVWqM2RqYPpU1aPxB8PBAkJqrfsRVjCHz5Y_X_7OXVIft-j-2UsLELzo7bLxMOwXwHSu9C8Eo_JBlBvq2H-2zwbT3wqR6S7M3-Az2I7guA_AUr6AP5</recordid><startdate>20150721</startdate><enddate>20150721</enddate><creator>Wang, Zhen</creator><creator>Zhao, Fei</creator><creator>Gao, Qian</creator><creator>Liu, Zhenlong</creator><creator>Zhang, Yongxin</creator><creator>Li, Xiaoyu</creator><creator>Li, Yuhuan</creator><creator>Ma, Weilie</creator><creator>Deng, Tao</creator><creator>Zhang, Zhizhen</creator><creator>Cen, Shan</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</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>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20150721</creationdate><title>Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication</title><author>Wang, Zhen ; Zhao, Fei ; Gao, Qian ; Liu, Zhenlong ; Zhang, Yongxin ; Li, Xiaoyu ; Li, Yuhuan ; Ma, Weilie ; Deng, Tao ; Zhang, Zhizhen ; Cen, Shan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c758t-1c44038c02fb05503ef356f8171233f6ec1134dd5007b10baa87d602ec851b403</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Analysis</topic><topic>Antiviral agents</topic><topic>Assaying</topic><topic>Biochemistry</topic><topic>Biotechnology</topic><topic>Cell Line, Tumor</topic><topic>Cell Survival</topic><topic>DNA-directed RNA polymerase</topic><topic>Drug development</topic><topic>Drug resistance</topic><topic>Drug Resistance, Viral - genetics</topic><topic>Drugs</topic><topic>Gaussia</topic><topic>Gene expression</topic><topic>Genes, Reporter</topic><topic>Genetic research</topic><topic>Genome</topic><topic>Genome, Viral</topic><topic>Genomes</topic><topic>Health risks</topic><topic>HEK293 Cells</topic><topic>High-throughput screening</topic><topic>Humans</topic><topic>Immunization</topic><topic>Influenza</topic><topic>Influenza A</topic><topic>Influenza A virus - genetics</topic><topic>Influenza A virus - physiology</topic><topic>Influenza, Human - virology</topic><topic>Inhibitors</topic><topic>Luciferase</topic><topic>Luciferases - metabolism</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Public health</topic><topic>Pyrrolidinones</topic><topic>Replication</topic><topic>Reproducibility</topic><topic>Reproducibility of Results</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA Replicase - genetics</topic><topic>RNA viruses</topic><topic>RNA, Small Interfering - metabolism</topic><topic>RNA, Viral - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Zhen</au><au>Zhao, Fei</au><au>Gao, Qian</au><au>Liu, Zhenlong</au><au>Zhang, Yongxin</au><au>Li, Xiaoyu</au><au>Li, Yuhuan</au><au>Ma, Weilie</au><au>Deng, Tao</au><au>Zhang, Zhizhen</au><au>Cen, Shan</au><au>Digard, Paul</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-07-21</date><risdate>2015</risdate><volume>10</volume><issue>7</issue><spage>e0133558</spage><epage>e0133558</epage><pages>e0133558-e0133558</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Influenza A virus (IAV) poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS) application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp) complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc) and blasticidin resistance gene (Bsd) were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>26196128</pmid><doi>10.1371/journal.pone.0133558</doi><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2015-07, Vol.10 (7), p.e0133558-e0133558 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1697759244 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS) Journals Open Access; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Analysis Antiviral agents Assaying Biochemistry Biotechnology Cell Line, Tumor Cell Survival DNA-directed RNA polymerase Drug development Drug resistance Drug Resistance, Viral - genetics Drugs Gaussia Gene expression Genes, Reporter Genetic research Genome Genome, Viral Genomes Health risks HEK293 Cells High-throughput screening Humans Immunization Influenza Influenza A Influenza A virus - genetics Influenza A virus - physiology Influenza, Human - virology Inhibitors Luciferase Luciferases - metabolism Plasmids Proteins Public health Pyrrolidinones Replication Reproducibility Reproducibility of Results Ribonucleic acid RNA RNA Replicase - genetics RNA viruses RNA, Small Interfering - metabolism RNA, Viral - genetics RNA-directed RNA polymerase Sensitivity analysis Statistical analysis Transcription Transcription (Genetics) Transcription, Genetic Viral infections Virology Virus replication Virus Replication - drug effects Viruses |
| title | Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-19T13%3A06%3A11IST&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=Establishment%20of%20a%20High-Throughput%20Assay%20to%20Monitor%20Influenza%20A%20Virus%20RNA%20Transcription%20and%20Replication&rft.jtitle=PloS%20one&rft.au=Wang,%20Zhen&rft.date=2015-07-21&rft.volume=10&rft.issue=7&rft.spage=e0133558&rft.epage=e0133558&rft.pages=e0133558-e0133558&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0133558&rft_dat=%3Cgale_plos_%3EA422565368%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=1697759244&rft_id=info:pmid/26196128&rft_galeid=A422565368&rft_doaj_id=oai_doaj_org_article_49c6fb6f272945598c099f97a27f9a5b&rfr_iscdi=true |