FIG 6.

FLuc expression from the dual-luciferase reporter is due to IRES activity. (A) BHK21 (left) and Vero E6 (right) cells were transfected with the dl 5′UTR DENV and ΔSV40 dl 5′UTR DENV plasmids (200 ng). The latter plasmid shares the full backbone of the dl 5′UTR DENV and lacks only the SV40 promoter. The RLuc (shaded bars) and FLuc (filled bars) activities obtained for the dl 5′UTR DENV DNA were set to 100%. (B) A 50 nM concentration of a scrambled control RNA (scRNA) or the siRLuc siRNA, which targets the RLuc ORF, was cotransfected with the dl 5′UTR DENV plasmid (200 ng) into BHK-21 (left) or Vero E6 (right) cells. RLuc (shaded bars) and FLuc (filled bars) activities were measured and are expressed relative to the luciferase activities obtained when cells were transfected with the scRNA, set at 100%. Statistical analysis was performed with a two-tailed t test. Asterisks indicate P values of <0.05; ns, no significant difference. The values shown are means (± standard errors of the means) for three independent experiments, each performed in duplicate.