The firefly and Gaussia luciferase genome segments are most likely competing for host and/or viral factors that are necessary for transcription and/or replication. To analyze whether a limiting availability of viral proteins is an important factor in the competition between firefly and Gaussia luciferase genome segments, we increased the amount of plasmids encoding the RNA polymerase subunits and NP in the transfection assay. Empty plasmid (pUC18) was included in the transfection mixture when needed to achieve the same total amount plasmid DNA for each transfection condition. Upon increasing the amounts of transfected plasmids encoding PB1/PB2/PA/NP, the normFluc/ Gluc ratio increased ,10-fold (Fig. 3A ) as a result of increased firefly luciferase expression levels ( Fig. S3A and B ). This result indicates that increased amounts of polymerase subunits and NP can alleviate the competition between the firefly and Gaussia luciferase genome segments. Increasing the amount of transfected NP-encoding plasmid alone did not affect the normFluc/Gluc ratio ( Fig. 3B ) or the absolute Fluc and Gluc levels ( Fig. S3C and D). Increasing the amount of polymerase subunit-encoding plasmids, but not of the NP-encoding plasmid, appeared to alleviate the competition between the two segments ( Fig. 3C ). However, it also negatively affected the reporter gene expression levels per se, with most dramatic effects being observed for the firefly luciferase reporter segment ( Fig. S3E and F). We speculate that this negative effect correlates with the requirement for NP for replication, which appears less stringent for short RNA templates [25, 26] . Although we did not analyze the NP and polymerase protein levels directly, our results indicate that the luciferase genome segments compete for RNA polymerase subunits and/or NP, with a limiting amount of polymerase subunits being the most likely explanation for the observed competition. However, we cannot exclude that the observed competition between reporter segments is partly caused by limiting amounts of host factors.