FIG 4.

Transcriptional activities of Bicis containing a short or a long IR. (A) Transcripts synthesized from Bicis in EBOV-infected cells. Huh-T7 cells were transfected with plasmid DNA carrying the indicated Bicis, along with a T7 RNA polymerase expression plasmid. At 12 h posttransfection, the cells were infected with EBOV at an MOI of 5 or left uninfected. Poly(A)⁺ mRNA was harvested at 48 h p.i. and analyzed by Northern hybridization with CAT-specific riboprobes. As a positive control, monocistronic minigenome 3E-5E encoding CAT was transfected (4). As a negative control, nontransfected cells infected with EBOV were used (lane 7). Shown is a representative result of three independent experiments. A short exposure (top) and a long exposure (bottom) of the X-ray film revealing readthrough mRNA are shown. The mRNA products are labeled as for Fig. 3B. (B) Transcripts synthesized in cells infected with Bici-containing viral supernatants. Supernatants of cells transfected and infected as described for panel A were collected at 72 h p.i. and used to infect Huh-T7 cells. At 48 h p.i., poly(A)⁺ mRNA was purified and analyzed as described for panel A. The experiment was repeated twice with similar results. (C) Transcriptional activities of Bicis in an EBOV minigenome system. BSR-T7/5 cells were transfected with Bici-carrying plasmids, along with expression plasmids for the EBOV polymerase complex. Cells were harvested at 48 h posttransfection and subjected to Northern hybridization. Shown is a representative result of four independent experiments.