Hexamer phasing governs transcription initiation in the 3′-leader of Ebola virus

Impact of hexamer phasing in the 3′-leader on transcription and replication. (A) Relative reporter activity of RC MGs with replacements of the NP (wt) HP as indicated (for structural details of leader variants, see Figs. 3, 4B). (B) As in panel A, but the same 3′-leader variants integrated into the RD MG scaffold. Mean values (±SEM), normalized to the native NP (wt) leader construct, are based on three biological replicates with two or three technical replicates each. Above each column, we calculated the activity (in %) of the individual mutant MG relative to the NP (wt) MG (=100%) after subtraction of the −L background from both. The diagrams in panels A and B combine results already shown in Figures 4 and 5. (C) Mean 2^−ΔC_T values obtained with RNA isolated from cells transfected with the NP (wt) MG to illustrate the abundance of viral mRNA (pink), cRNA (magenta), and vRNA (green), measured by a two-step strand-specific qRT-PCR of RC MG samples using the same cells as in panel A; the presence (+) or absence (−) of L is indicated below the x-axis. 2^−ΔC_T values were calculated for each experiment individually; mean 2^−ΔC_T values (±SEM) were derived from six to 16 independent experiments with three technical replicates each. 2^−ΔC_T values for mRNA were calculated as follows, based on the mean C_T (±SEM) values of 14.52 ± 0.15 for mRNA + cRNA, 20.97 ± 0.11 for cRNA, and 23.42 ± 0.09 for firefly luciferase mRNA: C_T (mRNA + cRNA) minus C_T (firefly luciferase mRNA), that is, 14.52 − 23.42 = −8.9, corresponding to a 2^−ΔC_T value of 477.7; C_T (cRNA) minus C_T (firefly luciferase mRNA), i.e. 20.97 − 23.42 = −2.45, corresponding to a 2^−ΔC_T value of 5.46; subtracting 5.46 from 477.7 then gives the 2^−ΔC_T value of 472.24 for mRNA. Above the cRNA and vRNA columns (+L), we calculated the mole percentage for cRNA and vRNA relative to mRNA (pink column) after subtraction of the corresponding −L background controls. For the binding sites of primer pairs, see Supplemental Figure S2. (D–F) Relative levels of viral mRNA, cRNA, and vRNA using the same cells as in panel A and normalized to the NP (wt) construct. Mean 2^−ΔΔC_T values (±SEM) were derived from at least three independent experiments with two or three technical replicates each. Dotted horizontal lines mark the level of the −L control. For experimental details, see Materials and Methods.