(A) Engineering of the spacer region to conform to hexamer phasing and (B) reverse engineering of the NP (wt) spacer region to violate hexamer phasing in RC MGs. (A) Luciferase reporter activities as a function of sequence/structure variation in the TSS/spacer region of MG 3′-leaders. Leaders containing the NP (wt) HP hairpin or native HP structures of internal EBOV genes instead are indicated by blue bars; HP structures violating hexamer phasing were engineered to restore the hexamer phase by deletion or insertion of nucleotides in the spacer region, either in the apical loop, stem, or between the stem and PE2 (gray bars). (B) Luciferase reporter activities of RC MGs carrying the NP (wt) HP or variants with 1-nt deletions or insertions, illustrated in the NP HP structure on the right (+: insertion; Δ: deletion) resulting in deviation from hexamer phasing. Values in (A) and (B) were normalized to the NP (wt) 3′-leader as control (100%). As a negative control (−L), the plasmid encoding the L gene was omitted in transfection of cells with the set of plasmids encoding EBOV proteins relevant to replication and transcription. Mean values (±SEM) are based on three independent experiments with at least three technical replicates each.
