Skip to main content
. 2014 Nov;88(21):12558–12571. doi: 10.1128/JVI.01863-14

FIG 5.

FIG 5

The length of the intergenic region regulates reinitiation efficiency at the VP30-VP24 gene border. (A) Scheme of mutant VP30/VP24 Bicis. Bici VP30/VP24-wt IR 144nt is depicted as in Fig. 3A. The complete sequence of the wild-type IR is shown underneath. The arrows mark nucleotides mutated to create a restriction site for cloning of Bici VP30/VP24 IR 293nt. The lines illustrate IR sequences in the respective truncation mutants. (B to G) Bicis containing the VP30-VP24 gene border were tested in the EBOV minigenome system and analyzed by Northern hybridization (B, D, F, and G) or by quantitative CAT assay (C and E). Shown are representative results of at least 3 independent experiments. The quantified CAT activity (+ standard deviation [SD]) shown in panels C and E was normalized to the Bici containing the wild-type gene border (wt IR 144nt; black bars). Expression levels from each Bici were compared to wild-type values using a one-sample t test (***, P < 0.001; **, P < 0.01; *, P < 0.05). (G) mRNA 2/mRNA 1 ratios of quantified Northern blots. mRNA ratios (+SD) of the truncation mutants (a representative blot is shown in panel F) were normalized to the Bici containing the wild-type gene border (wt IR 144nt; black bar). Expression levels for each Bici were compared to wild-type values using a one-sample t test (***, P < 0.001; **, P < 0.01; *, P < 0.05; no asterisk, not significant). (B and C) Deletion of the IR. (D and E) Elongation of the IR. (F and G) Truncation of the IR. The length of the IR is indicated for each mutant. CAM, chloramphenicol; ac CAM, acetylated CAM. Note that different exposure times were used for the blots shown in panels B and D, with the blot in panel D having the longer exposure time to show the presence of mRNA 2. (H) Analysis of the transcriptional activity of a Bici with an inhibitory IR in cells infected with EBOV. Huh-T7 cells were transfected with Bici DNA containing the wild-type VP30-VP24 gene border or a mutated gene border with a truncated IR (IR 20nt) and subsequently infected with EBOV as described in the legend to Fig. 4A. CAT-specific mRNA was analyzed by Northern hybridization. The experiment was repeated twice with the same outcome.