Subsequently, we analyzed to what extent reporter gene expression was affected by the presence of the natural viral genome segments. To this end, the reporter constructs were cotransfected with plasmids encoding each of the IAV genome segments under the same control of human RNA polymerase I promoter. As a control, empty plasmid (pUC18) was cotransfected. The different IAV segments significantly affected the firefly luciferase levels (Fig. 5A) . In general, the shorter segments gave stronger competition on the firefly expression compared to longer segments, with the M and NS segments having the largest effect. Correlation between inhibition on firefly luciferase expression and the gene length resulted in an R 2 value of 0.7 (Fig. 5C) , which indicates that 70% of the variance in firefly luciferase inhibition correlates with variability in genome segment length. In contrast, expression of Gaussia luciferase was hardly affected by the presence of the viral RNA segments (Fig. 5B) , while no correlation was observed between the modest decrease/increase of Gaussia luciferase expression and the genome segment length (Fig. S7) . We speculate that the lack of inhibition of Gaussia luciferase expression correlates with the very efficient replication/transcription of this reporter segment.