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Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identific...

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Autores principales: Fanunza, Elisa, Frau, Aldo, Sgarbanti, Marco, Orsatti, Roberto, Corona, Angela, Tramontano, Enzo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850405/
https://www.ncbi.nlm.nih.gov/pubmed/29495311
http://dx.doi.org/10.3390/v10020098
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author Fanunza, Elisa
Frau, Aldo
Sgarbanti, Marco
Orsatti, Roberto
Corona, Angela
Tramontano, Enzo
author_facet Fanunza, Elisa
Frau, Aldo
Sgarbanti, Marco
Orsatti, Roberto
Corona, Angela
Tramontano, Enzo
author_sort Fanunza, Elisa
collection PubMed
description The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.
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spelling pubmed-58504052018-03-16 Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling Fanunza, Elisa Frau, Aldo Sgarbanti, Marco Orsatti, Roberto Corona, Angela Tramontano, Enzo Viruses Article The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance. MDPI 2018-02-24 /pmc/articles/PMC5850405/ /pubmed/29495311 http://dx.doi.org/10.3390/v10020098 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fanunza, Elisa
Frau, Aldo
Sgarbanti, Marco
Orsatti, Roberto
Corona, Angela
Tramontano, Enzo
Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title_full Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title_fullStr Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title_full_unstemmed Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title_short Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
title_sort development and validation of a novel dual luciferase reporter gene assay to quantify ebola virus vp24 inhibition of ifn signaling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850405/
https://www.ncbi.nlm.nih.gov/pubmed/29495311
http://dx.doi.org/10.3390/v10020098
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