Next, we analyzed to what extent the competition between different reporter constructs is affected by the identity of the 39 and 59 UTRs. The genome segment UTRs provide signals for viral RNA transcription and replication, as well as for packaging of vRNP into virus particles [27] . The first 12 and 13 nucleotides at the 39 and 59 UTRs, which are highly conserved among the eight viral RNA segments, constitute the promoter structure for RNA synthesis [8, 9] . Also the non-conserved regions of the different segments have been implicated in viral RNA replication [28] . We inserted the 39 and 59 UTRs of the eight IAV-WSN segments (Table 1) into the extended Gaussia reporter plasmid and tested them in the competition assay with the firefly luciferase reporter construct FNP. The extended Gaussia construct was chosen instead of the short version as the former construct affects expression of the firefly luciferase construct less than the latter, resulting in a more balanced system in which it will be easier to detect UTRdependent up and down effects. Introduction of the UTRs of different segments into the extended Gaussia reporter construct affected the balance between the Gaussia and firefly luciferase expression to different extents (Fig. 4B) . Introduction of PB1, NA or NS segment UTRs resulted in balanced firefly and Gaussia luciferase expression levels as similar ratio's were observed when expressed alone or in combination (normFluc/Gluc ,1; Fig. 4B ). When the extended Gaussia construct was provided with the PB2, PA, HA and M segment UTRs, the normalized ratio's observed after co-transfection with FNP were similar to those observed when the construct containing the NP segment UTRs was used (normFluc/Gluc ,1), indicating that the balance had shifted in favor of Gaussia luciferase expression. Subsequently, we analyzed whether the absolute expression levels of the eight different Gaussia luciferase segments (Fig. S5A ) correlated with their ability to inhibit expression of firefly luciferase (Fig. S5B) . Correlation between inhibition of firefly luciferase expression and the expression of Gaussia luciferase with different UTRs resulted in an R 2 value of 0.8 (Fig. 4C) , which indicates that 80% of the variance in firefly luciferase inhibition correlates with variability in Gaussia luciferase expression levels. A similar R 2 value was obtained when the results obtained with the short GNP reporter segment were also taken into account (Fig. S6) . The results indicate that not only the expression of reporter genome segments, but also the balance between different reporter genome segments is affected by 39 and 59 UTRs, and that these two phenomena are largely correlated. Thus, Gaussia luciferase genome segments that are expressed to a higher extent are more efficient inhibitors of firefly luciferase expression driven by another genome segment.