FIG 2 .

Schematic overview of the reporter assay system and luciferase activity in QT6 cells. (A) The reporter plasmids contained the chicken RNA polymerase I promoter, mouse RNA polymerase I terminator, PR8 HA segment-derived NCR, and firefly luciferase gene. Linkers (28, 29, or 30 polynucleotides) that originated from the RNA sequence encoding amino acids across the HA cleavage site of ShimH5 were inserted between a start codon and the firefly luciferase gene lacking its start codon. The nucleotide positions different from those of the parental ShimH5 sequence are indicated in red. (B) QT6 cells were transfected with the reporter plasmid, pRL-TK Renilla luciferase transfection control reporter plasmid, and a mixture of PB2-, PB1-, PA-, and NP-expressing plasmids, and luciferase (firefly and Renilla luciferase) activities were measured. (C) In this assay, negative-sense vRNA templates are transcribed from the reporter plasmid by cellular RNA polymerase I, and then mRNA and cRNA are produced by the PR8 polymerases and NP, which are provided by the cotransfected protein expression plasmids. The transcripts containing 28- or 29-polynucleotide linkers produce mRNAs that are not in frame with the ORF of the reporter gene. Therefore, the firefly luciferase is expected to be expressed when nucleotides are inserted into the linker region of mRNA, cRNA, and/or vRNA to make the linker sequence in frame with the ORF of the reporter gene. The firefly luciferase activities were standardized using the values given by the activities of the transfection control, Renilla luciferase. (D and E) Luciferase activities were expressed relative to the empty plasmid and compared among the reporter plasmids containing the indicated linkers. Representative data from three independent experiments are shown. Relative luciferase activities are presented as the averages and standard deviations from triplicate wells. Statistical significance was calculated using Student’s t test (*, P < 0.05). Asterisks placed directly above bars indicate significant differences compared to the empty plasmid, and asterisks placed between bars show significant differences between the indicated bars.