Mutations in the 39UTR of the NP segment that increase gene expression have been described [23] . The nucleotide changes were predicted to improve base pairing of the 39 and 59 UTRs and thus to stabilize the panhandle structure. Considering the results described above, we expected that these mutations would affect competition between different reporter genome segments. The results show that reporter constructs containing these panhandlestabilizing UTRs (referred to as NPph; Fig. 6A ) indeed displayed 3-5 fold higher luciferase expression levels in the transfection assay than their wild-type UTR-containing counterparts (Fig. S8) . Remarkably, however, the normalized ratio between firefly and Gaussia luciferase was much more affected by the presence of the NPph UTRs. Introducing the NPph UTR in the background of the extended Gaussia construct (GFsNPph) resulted in a much decreased normFluc/Gluc when compared to its counterpart with the wild type NP UTRs (GFsNP; Fig. 6B) . A similar level of competition was not observed when the mutations that increase the number of base-pairs were introduced in the 59 UTR of the NP segment (referred to as NPphR; Fig. 6A ) instead of in the 39UTR. In this case, the Gaussia, rather than the firefly luciferase expression was affected by the co-transfection of both reporter plasmids (normFluc/Gluc .1; Fig. 6B and Fig. S8B ). Similar results were obtained when NPph UTR was introduced in the firefly luciferase genome segment (referred to as FNPph; Fig. 6C ). In general, normFluc/Gluc was increased when FNPph rather than FNP was used, except for the combination with GNPph ( Fig. 6C & Fig. S8C and D) . Thus, the balance between different segments is dramatically affected by the introduction of panhandle structure-stabilizing mutations in the 39UTR. Infection assay Neumann and Hobom (1995) previously reported increased reporter gene expression upon the introduction of panhandlestabilizing mutations in the 39 UTR. In their experimental system, however, the differences in reporter gene expression appeared much larger than ours. We hypothesized that this difference might be explained by Neuman and Hobom using virus infection to drive reporter gene expression, while we used transfection of polymerase subunit-and NP-encoding plasmids. Infection with IAV will not only provide viral RNA polymerase and NP, but will also introduce natural vRNPs that may compete with reporter genome segments for replication and/or transcription. Thus, in virusinfected cells, the natural virus genome segments might be preferentially replicated and transcribed over the reporter genome segments, unless the panhandle-stabilizing mutations in the 39 UTR are present. To test this hypothesis we compared reporter gene expression driven by co-transfection of expression plasmids (transfection assay) with expression driven by virus infection (infection assay). Reporter genes flanked either by natural NP UTRs or by the mutant NPph UTRs were used. Both firefly and Gaussia luciferase genes were expressed at high levels in the transfection assay, with the reporter constructs containing the NPph UTRs again displaying somewhat higher luciferase levels than their counterparts with the natural NP UTRs (Fig. 7A) . However, when using the infection assay, dramatic differences in reporter gene expression levels were observed (Fig. 7B) . Thus, while the reporter genes flanked by the NPph UTRs reached 1 to 2 fold higher expression levels than those flanked by the natural NP UTR in the transfection assay, this fold difference was much increased (130 to 160 fold) in the infection assay (Fig. 7C) . Quantitative RT PCR confirmed that mRNAs levels of the Gaussia luciferase RNAs were very similar, regardless of the presence of the natural NP UTRs or the mutant NPph UTRs in the transfection assay, but not in the infection assay (Fig. 7D) . These results are in agreement with our model, in which IAV genome segments compete for available resources, likely the viral proteins, to maximize their replication and/or transcription. Only reporter genome segments carrying panhandle-stabilizing mutations in their 39 UTR are able to efficiently compete with the natural genome segments in the infection assay. 