|Titolo:||Use of a Dual Luciferase Cell-Based Drug Screening Assay to Study VP24 Inhibition of JAK/STAT Pathway and its Reversion by Compounds|
CORONA, ANGELA (Penultimo)
TRAMONTANO, ENZO (Ultimo)
|Data di pubblicazione:||2018|
|Abstract:||Ebola virus has evolved several and diversified strategies to antagonize the interferon (IFN) response in target cells leading to a complete impairment of the innate immune system which contribute to the pathogenesis of infection. A decisive role is exerted by VP24 that disables cells to contrast viral replication and propagation by inhibiting the IFN system at level of JAK/STAT pathway. The early control of viremia is one of the keys for survival, hence, blocking an important determinant of virulence such as VP24 is a valuable subject for investigation and a crucial pharmacological target. No drugs currently have been approved for VP24. Based on these findings, driven by the effort to identify compounds able to inhibit VP24, we developed a new miniaturized dual drug screening assay to quantify the suppression of IFN cascade by VP24 and the effect of potential inhibitors. The assay is based on the transient cotransfection of HEK293T cells with a luciferase reporter under the control of the promoter of IFN stimulated genes (pISRE-luc), a plasmid expressing VP24 and RL-TK, as control of transfection efficiency. The stimulation with IFN- led to the activation of ISRE expression in cells transfected only with pISRE-luc. In contrast, cotransfection with VP24 led to the inhibition of ISRE transcription. Addition of compounds, such the same IFN- , led to the partial restoration of the pathway. We optimized the assay to achieve excellent signal and robust performance. Further, the normalization with a Renilla luciferase control allowed to minimize variability between experiments providing high reproducibility.|
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