Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Reagents
2.2. Construction of EBOV VP24 Mammalian Expression Plasmid
2.3. Luciferase Reporter Gene Assay
2.4. EBOV VP24 Inhibition Assay
2.5. Immunoblot Analysis
2.6. Data Analysis
3. Results
3.1. Establishment of a Miniaturized Cell-Based Assay for Evaluating VP24 Inhibition of JAK/STAT Cascade Activation
3.2. Optimization of Stimulation Conditions and Evaluation of VP24 IC50
3.3. Luciferase Assay Signal Stability
3.4. Assay Validation
3.5. Designing the Drug Screening Assay
3.6. IFN-α Partially Reverts VP24 Inhibition of JAK/STAT Cascade
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Signal | Coefficients of Variation, CV (%) |
---|---|
Min | 6.1 |
Mid | 4.6 |
Max | 3.3 |
Inter-Plate Test | pISRE-luc Activation (%) | Inter-Day Test | pISRE-luc Activation (%) |
---|---|---|---|
Plate 1 | 52.4 | Plate 1 | 49.3 |
Plate 2 | 50.8 | Plate 2 | 49.0 |
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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. https://doi.org/10.3390/v10020098
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(2):98. https://doi.org/10.3390/v10020098
Chicago/Turabian StyleFanunza, Elisa, Aldo Frau, Marco Sgarbanti, Roberto Orsatti, Angela Corona, and Enzo Tramontano. 2018. "Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling" Viruses 10, no. 2: 98. https://doi.org/10.3390/v10020098
APA StyleFanunza, E., Frau, A., Sgarbanti, M., Orsatti, R., Corona, A., & Tramontano, E. (2018). Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling. Viruses, 10(2), 98. https://doi.org/10.3390/v10020098