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Fig. 1 | Genome Medicine

Fig. 1

From: A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle

Fig. 1

Minigenome optimization. a Graphical representation of the EBOV minigenome assay and structure of the minigenome. A minigenome consisting of a reporter open reading frame (rep) flanked by the EBOV non-coding leader (ldr) and trailer (trl) regions under the control of a T7 promoter (T7p) and terminator (T7t) is expressed in mammalian cells. An HDV-ribozyme (HDV) ensures an authentic 3′ end of the minigenome. T7 polymerase (T7), which initially transcribes the minigenome, and the EBOV proteins NP, VP35 (35), VP30 (30) and L, which are necessary for replication and transcription of the minigenome, are produced from RNA polymerase II-driven expression vectors. Firefly luciferase (FF) is expressed from an additional RNA polymerase II-driven expression plasmid and serves as a transfection/cell viability control, and is used to normalize minigenome reporter activity. b Reporter activity from minigenomes expressing various reporters. Minigenomes encoding various reporters, either Renilla luciferase (hrLuc), nano-luciferase (nLuc) or a nano-luciferase with an attached PEST-sequence (nLuc-PEST), were tested in the assay shown in panel a. The viral polymerase was substituted for empty vector in duplicate samples to establish the background reporter activity for each construct. Nano-luciferase and Renilla luciferase values were normalized to firefly luciferase values. Means and standard deviations of 28 biological replicates from 3 independent experiments in 96-well format are shown

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