Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection
Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepa...
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| Format: | Article |
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MDPI AG
2020-06-01
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| Series: | Proceedings |
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| Online Access: | https://www.mdpi.com/2504-3900/50/1/45 |
| _version_ | 1797565348119052288 |
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| author | Benedita K. L. Feron Joachim J. Bugert Simon C. W. Richardson |
| author_facet | Benedita K. L. Feron Joachim J. Bugert Simon C. W. Richardson |
| author_sort | Benedita K. L. Feron |
| collection | DOAJ |
| description | Here, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (<i>p</i> = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (<i>p</i> = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant <i>Bacillus </i><i>anthracis</i> PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of <i>B.</i><i> </i><i>anthracis</i> lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (<i>p</i> = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (<i>p</i> = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the Pierce<sup>TM</sup> Firefly Luciferase Glow Assay Kit by Thermo Scientific<sup>TM</sup>. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models. |
| first_indexed | 2024-03-10T19:10:53Z |
| format | Article |
| id | doaj.art-282e25915ca74c69a64a0b7b2e12b402 |
| institution | Directory Open Access Journal |
| issn | 2504-3900 |
| language | English |
| last_indexed | 2024-03-10T19:10:53Z |
| publishDate | 2020-06-01 |
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| spelling | doaj.art-282e25915ca74c69a64a0b7b2e12b4022023-11-20T03:45:34ZengMDPI AGProceedings2504-39002020-06-015014510.3390/proceedings2020050045Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) InfectionBenedita K. L. Feron0Joachim J. Bugert1Simon C. W. Richardson2The Exogenix Laboratory, Faculty of Engineering and Science, University of Greenwich, Chatham ME4 4TB, UKInstitut für Mikrobiologie der Bundeswehr, 80937 Munich, GermanyThe Exogenix Laboratory, Faculty of Engineering and Science, University of Greenwich, Chatham ME4 4TB, UKHere, we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) infection. siRNAs were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilized. After the electroporation of 100 nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3% ± 7.4% (SEM: n = 4) (<i>p</i> = 0.0004) relative to the cells treated only with the virus (33.9% ± 12%, SEM: n = 4). Furthermore, 100 nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1% ± 11.2% cell viability (SEM: n = 4) relative to the control cells treated with ZIKV (<i>p</i> = 0.0021). The cells were electroporated with siRNA prior to ZIKV infection and viability was monitored four days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant <i>Bacillus </i><i>anthracis</i> PA83 (octomer-forming mutants), co-incubated with the N-terminal 255 amino acids of <i>B.</i><i> </i><i>anthracis</i> lethal factor (LFn) fused in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50 µg/mL (each). Here, baby hamster kidney (BHK) cells, treated with 100 nM siRNA Feron Zv-1, yielded 79.0% ± 4.0% viability relative to the control (50.2% ± 1.7%, SEM: n = 3) three days after exposure to ZIKV (<i>p</i> = 0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123% ± 46% (SEM: n = 18), relative to 8% ± 16% (SEM: n = 18) for the same concentration of control HeLa exosomes, was recorded (<i>p</i> = 0.0416). In each instance, 0.3 moI was used and cell viability monitored using the Pierce<sup>TM</sup> Firefly Luciferase Glow Assay Kit by Thermo Scientific<sup>TM</sup>. Here, we show for the first time that siRNA can significantly reduce ZIKV-induced cell killing. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment, as well as testing the siRNAs and delivery systems within more complex models.https://www.mdpi.com/2504-3900/50/1/45Zika virussiRNAexosomeanthrax toxin |
| spellingShingle | Benedita K. L. Feron Joachim J. Bugert Simon C. W. Richardson Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection Proceedings Zika virus siRNA exosome anthrax toxin |
| title | Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection |
| title_full | Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection |
| title_fullStr | Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection |
| title_full_unstemmed | Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection |
| title_short | Pioneering siRNA-Mediated Protection of Mammalian Cells against Zika Virus (MR-766) Infection |
| title_sort | pioneering sirna mediated protection of mammalian cells against zika virus mr 766 infection |
| topic | Zika virus siRNA exosome anthrax toxin |
| url | https://www.mdpi.com/2504-3900/50/1/45 |
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