FIG 4.
Role of VP24 in regulating genome replication and transcription. (A) Influence of minigenome-expressed VP24 on replication and transcription in a tetracistronic minigenome assay. 293 cells were transfected with expression plasmids encoding tetracistronic minigenomes containing a wild-type VP24 gene (VP24-WT) or a VP24 gene in which 3 stop codons were inserted immediately after the start codon (VP24–3×-stop), abolishing expression of VP24; expression plasmids encoding the Ebola virus RNP proteins NP, VP35, VP30, and L; and an accessory expression plasmid encoding T7 RNA polymerase. As a negative control, the expression plasmid for L was omitted (−L). After 72 h, reporter activity was determined. The means and standard deviations from 7 independent experiments are shown. (B) Influence of plasmid-expressed VP24 on replication and transcription in a tetracistronic minigenome assay. Minigenome assays using the tetracistronic minigenome with the VP24 gene in which 3 stop codons were inserted immediately after the start codon were performed as described in the legend to panel A. In addition, the indicated amounts of pCAGGS-VP24 were cotransfected. The means and standard deviations for 6 biological replicates from 2 independent experiments are shown. (C) Efficacy of miRNA-mediated knockdown of VP24. 293 cells expressing Tim1 and miRNAs against Ebola virus VP24 or GFP were infected with wild-type Ebola virus at an MOI of 1. At 24 h after infection, cells were lysed and lysates were analyzed by Western blotting using antibodies against NP or VP24. (D) Effect of miRNAs on virus replication. 293 cells expressing Tim1 and miRNAs against Ebola virus (EBOV) L (anti-L), Ebola virus VP24 (anti-VP24), or an unrelated protein (anti-luc2) were infected with a recombinant Ebola virus expressing GFP at an MOI of 0.1. At 4 days after infection, cells were visualized by fluorescence microscopy. Random fields are shown. (E) Influence of VP24 on genome replication and transcription in infection. 293 cells were transfected with expression plasmids encoding Tim1 and miRNAs directed against VP24 (anti-VP24), L (anti-L), or an unrelated protein (anti-GFP). After 24 h, cells were infected with a recombinant Ebola virus expressing luciferase at an MOI of 3. Reporter activity in cells was determined every 1 to 2 h for 32 h after infection. The means and standard deviations for 6 biological replicates from 2 independent experiments are shown as absolute values. (F) Relative influence of VP24 on genome replication and transcription in infection. The data from panel E are expressed relative to the reporter activity observed in cells transfected with an expression plasmid for an unrelated protein. *, significant differences (P < 0.05) in reporter activity compared to that for control cells.