Titolo:  Development and validation of a novel dual luciferase reporter gene assay to quantify Ebola virus VP24 inhibition of IFN signaling
Data di pubblicazione:  2018
Data di prima pubblicazione on-line:  2018
Autori:  Fanunza, Elisa; Frau, Aldo; Sgarbanti, Marco; Orsatti, Roberto; Corona, Angela; Tramontano, Enzo
Numero degli autori:  6
Lingua:  Inglese
Presenza coautori internazionali:  no
Rivista:  VIRUSES
Volume:  10
Fascicolo:  2
Numero di pagine:  12
Digital Object Identifier (DOI):  http://dx.doi.org/10.3390/v10020098
Codice identificativo Pubmed:  29495311
Codice identificativo Scopus:  2-s2.0-85042770141
Codice identificativo ISI:  WOS:000427544100048
URL:  http://www.mdpi.com/1999-4915/10/2/98
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.
Parole chiave:  Ebola virus VP24; IFN inhibition; IFN signaling; Drug development; Dual luciferase gene reporter assay; Innate immunity
Tipologia: 1.1 Articolo in rivista

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