A cell-based screening system for anti-influenza A virus agents
Identifieur interne : 000430 ( Pmc/Checkpoint ); précédent : 000429; suivant : 000431A cell-based screening system for anti-influenza A virus agents
Auteurs : Wan Ying Wong [Malaisie] ; Sheng Wei Loh [Malaisie] ; Wei Lun Ng [Malaisie] ; Ming Cheang Tan [Malaisie] ; Kok Siong Yeo [Malaisie] ; Chung Yeng Looi [Malaisie] ; Mohd Jamil Maah [Malaisie] ; Chee-Kwee Ea [Malaisie]Source :
- Scientific Reports [ 2045-2322 ] ; 2015.
Abstract
Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity
Url:
DOI: 10.1038/srep08672
PubMed: 25728279
PubMed Central: 4345322
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A cell-based screening system for anti-influenza A virus agents</title><author><name sortKey="Wong, Wan Ying" sort="Wong, Wan Ying" uniqKey="Wong W" first="Wan Ying" last="Wong">Wan Ying Wong</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Loh, Sheng Wei" sort="Loh, Sheng Wei" uniqKey="Loh S" first="Sheng Wei" last="Loh">Sheng Wei Loh</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ng, Wei Lun" sort="Ng, Wei Lun" uniqKey="Ng W" first="Wei Lun" last="Ng">Wei Lun Ng</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Tan, Ming Cheang" sort="Tan, Ming Cheang" uniqKey="Tan M" first="Ming Cheang" last="Tan">Ming Cheang Tan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yeo, Kok Siong" sort="Yeo, Kok Siong" uniqKey="Yeo K" first="Kok Siong" last="Yeo">Kok Siong Yeo</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Looi, Chung Yeng" sort="Looi, Chung Yeng" uniqKey="Looi C" first="Chung Yeng" last="Looi">Chung Yeng Looi</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Pharmacology, Faculty of Medicine, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Maah, Mohd Jamil" sort="Maah, Mohd Jamil" uniqKey="Maah M" first="Mohd Jamil" last="Maah">Mohd Jamil Maah</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Department of Chemistry, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ea, Chee Kwee" sort="Ea, Chee Kwee" uniqKey="Ea C" first="Chee-Kwee" last="Ea">Chee-Kwee Ea</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25728279</idno><idno type="pmc">4345322</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345322</idno><idno type="RBID">PMC:4345322</idno><idno type="doi">10.1038/srep08672</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000097</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000097</idno><idno type="wicri:Area/Pmc/Curation">000097</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000097</idno><idno type="wicri:Area/Pmc/Checkpoint">000430</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000430</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A cell-based screening system for anti-influenza A virus agents</title><author><name sortKey="Wong, Wan Ying" sort="Wong, Wan Ying" uniqKey="Wong W" first="Wan Ying" last="Wong">Wan Ying Wong</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Loh, Sheng Wei" sort="Loh, Sheng Wei" uniqKey="Loh S" first="Sheng Wei" last="Loh">Sheng Wei Loh</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ng, Wei Lun" sort="Ng, Wei Lun" uniqKey="Ng W" first="Wei Lun" last="Ng">Wei Lun Ng</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Tan, Ming Cheang" sort="Tan, Ming Cheang" uniqKey="Tan M" first="Ming Cheang" last="Tan">Ming Cheang Tan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Yeo, Kok Siong" sort="Yeo, Kok Siong" uniqKey="Yeo K" first="Kok Siong" last="Yeo">Kok Siong Yeo</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Looi, Chung Yeng" sort="Looi, Chung Yeng" uniqKey="Looi C" first="Chung Yeng" last="Looi">Chung Yeng Looi</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Pharmacology, Faculty of Medicine, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Maah, Mohd Jamil" sort="Maah, Mohd Jamil" uniqKey="Maah M" first="Mohd Jamil" last="Maah">Mohd Jamil Maah</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Department of Chemistry, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ea, Chee Kwee" sort="Ea, Chee Kwee" uniqKey="Ea C" first="Chee-Kwee" last="Ea">Chee-Kwee Ea</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></nlm:aff><country xml:lang="fr">Malaisie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity <italic>in vivo</italic> and <italic>in vitro</italic>. NiPT5 impedes the signaling cascades that lead to the activation of NF-κB in response to different stimuli, such as LPS and TNFα. Using our cell-based screening system, we report that pretreating cells with NiPT5 protects cells from influenza A virus (IAV) and vesicular stomatitis virus (VSV) infection. Furthermore, NiPT5 inhibits replication of IAV by inhibiting transcription and translation of vRNAs of IAV. Additionally, NiPT5 reduces IAV-induced type I interferon response and cytokines production. Moreover, NiPT5 prevents activation of NF-κB, and IRF3 in response to IAV infection. These results demonstrate that NiPT5 is a potent antiviral agent that inhibits the early phase of IAV replication.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Samji, T" uniqKey="Samji T">T. Samji</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Szatraj, K" uniqKey="Szatraj K">K. Szatraj</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ison, M G" uniqKey="Ison M">M. G. Ison</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bavin, E M" uniqKey="Bavin E">E. M. Bavin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hamre, D" uniqKey="Hamre D">D. Hamre</name></author><author><name sortKey="Brownlee, K A" uniqKey="Brownlee K">K. A. Brownlee</name></author><author><name sortKey="Donovick, R" uniqKey="Donovick R">R. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25728279</article-id><article-id pub-id-type="pmc">4345322</article-id><article-id pub-id-type="pii">srep08672</article-id><article-id pub-id-type="doi">10.1038/srep08672</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A cell-based screening system for anti-influenza A virus agents</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Wong</surname><given-names>Wan Ying</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Loh</surname><given-names>Sheng Wei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ng</surname><given-names>Wei Lun</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Tan</surname><given-names>Ming Cheang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yeo</surname><given-names>Kok Siong</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Looi</surname><given-names>Chung Yeng</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Maah</surname><given-names>Mohd Jamil</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Ea</surname><given-names>Chee-Kwee</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Institute of Biological Sciences, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></aff><aff id="a2"><label>2</label><institution>Department of Pharmacology, Faculty of Medicine, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></aff><aff id="a3"><label>3</label><institution>Department of Chemistry, Faculty of Science, University of Malaya</institution>, 50603 Kuala Lumpur,<country>Malaysia</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>eacheekwee@um.edu.my</email></corresp></author-notes><pub-date pub-type="epub"><day>02</day><month>03</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>5</volume><elocation-id>8672</elocation-id><history><date date-type="received"><day>16</day><month>10</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>01</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015, Macmillan Publishers Limited. All rights reserved</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Macmillan Publishers Limited. All rights reserved</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>Emerging of drug resistant influenza A virus (IAV) has been a big challenge for anti-IAV therapy. In this study, we describe a relatively easy and safe cell-based screening system for anti-IAV replication inhibitors using a non-replicative strain of IAV. A nickel (II) complex of polyhydroxybenzaldehyde N4-thiosemicarbazone (NiPT5) was recently found to exhibit anti-inflammatory activity <italic>in vivo</italic> and <italic>in vitro</italic>. NiPT5 impedes the signaling cascades that lead to the activation of NF-κB in response to different stimuli, such as LPS and TNFα. Using our cell-based screening system, we report that pretreating cells with NiPT5 protects cells from influenza A virus (IAV) and vesicular stomatitis virus (VSV) infection. Furthermore, NiPT5 inhibits replication of IAV by inhibiting transcription and translation of vRNAs of IAV. Additionally, NiPT5 reduces IAV-induced type I interferon response and cytokines production. Moreover, NiPT5 prevents activation of NF-κB, and IRF3 in response to IAV infection. These results demonstrate that NiPT5 is a potent antiviral agent that inhibits the early phase of IAV replication.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>A cell-based screening system for IAV replication inhibitors.</title><p>(a) PB1Flank-eGFP viral RNA contains the 80 terminal coding nucleotides with mutated start codon flanking by untranslated regions from the PB1 segment. (b) A549-PB1 cells were seeded in a 96 wells plate and infected with different MOI of PR8-PB1flank-eGFP virus (IAV). After 24 hours, cells were stained with ER tracker as an internal control to normalize the eGFP fluorescent signal. The total fluorescent signals were measured with a Tecan Infinite F200 Pro fluorometer (b). Results represent the mean ± SD in quadruplicate experiments. Cells were visually examined on an Olympus IX73 inverted microscope at 200× final magnification and photographed using an Olympus DP73 digital camera and Cellsens standard software (c); or analyzed with FACS (d). Scale: 20 μm.</p></caption><graphic xlink:href="srep08672-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>NiPT5 inhibits IAV infection.</title><p>(a) A549-PB1 cells were cultured with various concentrations of NiTP5 as indicated. After two days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments. (b) A549-PB1 cells were seeded in a 96 wells plate. A549-PB1 cells were pre-treated with NiPT5 at various concentrations for 4 hours prior infecting the cells with PR8-PB1flank-eGFP virus (IAV) at a MOI 1.0. After 24 hours, cells were stained with ER tracker and analyzed as in <xref ref-type="fig" rid="f1">Fig. 1</xref>, microscopy (b); fluorometry (c). (d–e) A549-PB1 cells were treated as in (b–c), except that VSV-GFP virus was used for infection. Results represent the mean ± SD in quadruplicate experiments. Student's t-test: *, p < 0.05; **, p < 0.01. Scale: 20 μm.</p></caption><graphic xlink:href="srep08672-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>NiPT5 prevents the replication of influenza virus.</title><p>(a) A549-PB1 cells were treated with various concentrations of NiPT5 as indicated four hours prior to infecting the cells with PR8-PB1flank-eGFP virus (IAV) at a MOI 1.0. After twelve hours, the expressions of virus NP, M1 and NS1 were measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. (b) The A549-PB1 cells were treated as in (a), whole cell extracts were prepared and were subjected to immunoblotting with antibodies against NP and HSP90. Full-length blot is presented in <xref ref-type="supplementary-material" rid="s1">Supplementary Figure S4a</xref>. (c) A549-PB1 cells were pre-treated with 10 μM NiPT5 for four hours prior to infecting the cells with IAV virus at a MOI 0.5. After twenty hours, infected cells were analyzed with FACS (left panel) while the supernatant was harvested and subjected to viral titering assay using FACS (right panel). IP: infectious viral particle. Student's t-test: **, p < 0.01.</p></caption><graphic xlink:href="srep08672-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>NiPT5 inhibits IAV induced ISGs and cytokines expression.</title><p>(a) A549-PB1 cells were infected with PR8-PB1flank-eGFP virus (IAV) at a MOI 1.0. The expression of vRNAs, ISGs and cytokines was measured with RT-qPCR at the indicated time periods. (b) A549-PB1 cells were treated with various concentrations of NiPT5 as indicated four hours prior to infecting the cells with IAV at a MOI 1.0. After twelve hours, the expression of various ISGs and cytokines was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p < 0.05; **: p < 0.01. (c–d) Whole cell extracts were prepared from A549-PB1 cells as in (b), and examined by immunoblotting using antibodies against p-IκBα, Rel. Intensity: intensity of bands was quantified using the Image Lab (BioRad) software and was normalized to actin (c), p-IRF3 (d). Full-length blots are presented in <xref ref-type="supplementary-material" rid="s1">Supplementary Figure S4</xref>.</p></caption><graphic xlink:href="srep08672-f4"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Malaisie</li></country></list><tree><country name="Malaisie"><noRegion><name sortKey="Wong, Wan Ying" sort="Wong, Wan Ying" uniqKey="Wong W" first="Wan Ying" last="Wong">Wan Ying Wong</name></noRegion><name sortKey="Ea, Chee Kwee" sort="Ea, Chee Kwee" uniqKey="Ea C" first="Chee-Kwee" last="Ea">Chee-Kwee Ea</name><name sortKey="Loh, Sheng Wei" sort="Loh, Sheng Wei" uniqKey="Loh S" first="Sheng Wei" last="Loh">Sheng Wei Loh</name><name sortKey="Looi, Chung Yeng" sort="Looi, Chung Yeng" uniqKey="Looi C" first="Chung Yeng" last="Looi">Chung Yeng Looi</name><name sortKey="Maah, Mohd Jamil" sort="Maah, Mohd Jamil" uniqKey="Maah M" first="Mohd Jamil" last="Maah">Mohd Jamil Maah</name><name sortKey="Ng, Wei Lun" sort="Ng, Wei Lun" uniqKey="Ng W" first="Wei Lun" last="Ng">Wei Lun Ng</name><name sortKey="Tan, Ming Cheang" sort="Tan, Ming Cheang" uniqKey="Tan M" first="Ming Cheang" last="Tan">Ming Cheang Tan</name><name sortKey="Yeo, Kok Siong" sort="Yeo, Kok Siong" uniqKey="Yeo K" first="Kok Siong" last="Yeo">Kok Siong Yeo</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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