(A) Schematic representation of the RC MG used in the presented study. The sequence complementary to the mRNA encoding the Renilla luciferase reporter mRNA is indicated by the gray box; the red segment corresponds to the hairpin structure shown at the top (TSS plus spacer sequence); white boxes mark sequences antisense (as) to the 5′-UTR of the NP mRNA and antisense to the 3′-UTR of the L mRNA. (B) Relative reporter activity of RC MGs in which the wild type (wt) NP hairpin was replaced with hairpin structures (see Figs. 1A, 3 for details) derived from the transcription start region of internal EBOV genes. Data (±standard error of the mean, SEM), normalized to the native NP (wt) construct, are based on at least three biological replicates with two or three technical replicates each. (C) Schematic representation of the 3′-leader promoter region depicting an extended UN5 hexamer pattern from PE2 to position −51 in PE1 with a single interruption at G−75, thus defining a reference point (nt −51) in PE1 and linking UN5 hexamer phasing between PE1 and PE2. Accordingly, the distance between nt −51 and −80 is 5 × 6 = 30 nt in the wt MG (light gray box on the left). Only the mutant construct with the VP40 HP maintained a hexamer phasing (66 nt) between PE1 and PE2, whereas mutant MG constructs harboring the VP35, GP, VP30, VP24, or L HP, respectively, resulted in spacings not divisible by 6 (light gray box on the right). For color coding of the sequence, see legend to Figure 1B.
