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

Viruses
Elisa FanunzaEnzo Tramontano

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 parameter...Continue Reading

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Citations

Apr 17, 2020·The Journal of Dairy Research·Xubin LuZhangping Yang
May 6, 2020·Antimicrobial Agents and Chemotherapy·Elisa FanunzaEnzo Tramontano
Aug 17, 2019·Expert Opinion on Drug Discovery·Rafael Ferreira DantasFloriano Paes Silva-Junior
Nov 21, 2019·Antiviral Chemistry & Chemotherapy·Francesco Di PalmaPaolo Ruggerone

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Methods Mentioned

BETA
Transfection
electrophoresis
Assay
ELISA
isothermal
pull down
X-ray

Software Mentioned

GraphPad Prism

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