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Viruses 2018, 10(2), 98; https://doi.org/10.3390/v10020098

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

1
Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy
2
Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy
3
Genetics and Biomedical Research Institute, National Research Council, 09042 Monserrato, Italy
*
Author to whom correspondence should be addressed.
Received: 11 January 2018 / Revised: 16 February 2018 / Accepted: 22 February 2018 / Published: 24 February 2018
(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)
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Abstract

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. View Full-Text
Keywords: Ebola virus VP24; innate immunity; IFN signaling; IFN inhibition; dual luciferase gene reporter assay; drug development Ebola virus VP24; innate immunity; IFN signaling; IFN inhibition; dual luciferase gene reporter assay; drug development
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Fanunza, E.; Frau, A.; Sgarbanti, M.; Orsatti, R.; Corona, A.; Tramontano, E. Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling. Viruses 2018, 10, 98.

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