Author Contributions Competition between Influenza A Virus Genome Segments Influenza A virus (IAV) contains a segmented negative-strand RNA genome. How IAV balances the replication and transcription of its multiple genome segments is not understood. We developed a dual competition assay based on the co-transfection of firefly or Gaussia luciferase-encoding genome segments together with plasmids encoding IAV polymerase subunits and nucleoprotein. At limiting amounts of polymerase subunits, expression of the firefly luciferase segment was negatively affected by the presence of its Gaussia luciferase counterpart, indicative of competition between reporter genome segments. This competition could be relieved by increasing or decreasing the relative amounts of firefly or Gaussia reporter segment, respectively. The balance between the luciferase expression levels was also affected by the identity of the untranslated regions (UTRs) as well as segment length. In general it appeared that genome segments displaying inherent higher expression levels were more efficient competitors of another segment. When natural genome segments were tested for their ability to suppress reporter gene expression, shorter genome segments generally reduced firefly luciferase expression to a larger extent, with the M and NS segments having the largest effect. The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells. Our data indicate that IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription. The mechanism of replication and transcription varies greatly among viruses depending on the nature and structure of their viral genomes. Negative-strand RNA viruses replicate their viral genome via the synthesis of full length positive-strand complementary RNA (cRNA) molecules that in turn serve as templates for the synthesis of negative-strand virion RNA (vRNA) genomes. The negative-strand genomes also function as templates for the production of mRNAs [1, 2] . In non-segmented negative-strand RNA viruses, sequential transcription of successive genes results in a gradient of transcript abundance that steadily decreases towards the end of the template. Thus, the expression level of each gene is governed by the gene order [3] . This does, however, not apply to all negative-strand viruses as some of them acquired segmented genomes during their evolution. Each genome segment of these viruses is individually replicated and transcribed, necessitating careful regulation of these distinctive processes to generate sufficient vRNAs and proteins for the production of progeny virions [2] .