To get more insight in the mechanism by which IAV regulates and balances the replication and transcription of its genome segments, we converted the IAV single reporter assay into a dual reporter assay, by which the expression of two different luciferase genes can be monitored simultaneously. This assay more closely resembles the multiple segment transcription and replication conditions that occur in IAV infected cells than the single reporter assay. Our results indicate that different vRNA segments compete with each other, as transcription/replication of one vRNA Figure 1 . Schematic representations of the dual luciferase reporter constructs, and of transfection and infection assays. A) Schematic outline of the firefly and Gaussia luciferase reporter constructs. The firefly and Gaussia luciferase genes, flanked by 39 and 59 UTR of the NP segment, were inserted in antisense orientation between a PolI promoter and a ribozyme sequence, resulting in FNP and GNP, respectively. The extended Gaussia luciferase reporter construct (GFsNP) additionally contains the 39 terminal half of the firefly luciferase gene (indicated as Fs) behind the stop codon of the Gaussia gene. B) HEK 293T cells were transfected with one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases (PB1, PB2, PA) and NP either by simultaneous co-transfection of expression plasmids (transfection assay) or by infection with IAV at an MOI 1 at 24 h post-transfection (infection assay). The firefly and Gaussia luciferase expression levels can be measured consecutively using a dual luciferase assay system (Promega) 24 h post-transfection or post-infection. doi:10.1371/journal.pone.0047529.g001 segment can affect that of another. Using this multiple segment reporter assay we subsequently assessed the contribution of vRNA segment length, UTRs sequence and coding sequence to the competition between the different segments.