IRES-mediated translation in A549 cells infected with recombinant adenoviruses. (A) Schematic of bicistronic reporter DNA. The arrow indicates the transcription-initiation site of the murine cytomegalovirus immediate early promoter. Firefly luciferase and Renilla luciferase have independent translation initiation and termination codons. SV40 An, simian virus 40 polyadenylation signal. (B) IRES activity in cells infected with adenovirus encoding bicistronic mRNA. The x axis indicates IRES encoded in the recombinant adenovirus. PV 5′NC-X472, poliovirus IRES with XhoI linker substitution at nucleotide 472; HCV Ø28_69, HCV IRES with deletion of nucleotides 28_69. (C) Northern blot hybridization of A549 cells. Cells were infected with recombinant adenoviruses indicated at the top. RNA size markers are given at left. Lane 1, monocistronic firefly luciferase; lane 2, monocistronic Renilla luciferase; lane 3, bicistronic mRNA, no IRES; lane 4, bicistronic mRNA with P3/119/70 IRES; lane 5, bicistronic mRNA with P3/Sabin IRES; lane 6, bicistronic mRNA with HCV IRES; lane 7, bicistronic mRNA with HCV IRES lacking nucleotides 28_69. First two lanes are from the same gel as lanes 3_7 but were exposed for a longer period of time. (D) Luciferase expression in adenovirus-infected cells. Upper panel: Firefly luciferase (white bars) and Renilla luciferase expression (black bars). Luciferase was normalized to the P3/119/70 IRES value for each reporter protein, and also to the quantity of bicistronic mRNA determined by Northern blot hybridization. Lower panels: Representative Northern blot hybridizations of RNA from the same infected cells. IRES encoded by bicistronic mRNA is indicated at the bottom. Data points in B and D, the results of separate experiments, are the mean of three infections, and error bars indicate SD.