Although the above data were consistent with reduced delivery of the vRNA by the infecting viruses, we also considered the possibility that the AUG1/2 Kozak mutations perturbed the function of the viral RNA promoter (either the 3 0 -end of vRNA or the 5 0 -end of cRNA). This could lead to a reduction in segment 2 vRNA levels with a potential secondary effect of reducing the quantity available to be packaged into virions. Although the mutations lie well outside of the conserved promoter region, there are precedents for sequence alterations in the non-unique regions of a segment affecting RNA synthesis (30, 43, 44) . To examine this possibility in isolation, the amount of segment 2 produced from RNPs reconstituted by transfection was measured. Wild-type PB2, PB1, PA and NP were transfected into cells with the reverse genetics plasmids encoding the mutant segment 2 vRNAs. The segment 2 plasmids would be transcribed by RNA Polymerase I to produce a negative sense segment 2 transcript that would be encapsidated, transcribed and replicated by the WT RNP proteins. In addition, mutant PB1 protein would be also be expressed from the vRNAs with non synonymous changes to the PB1 gene (G28A, T32G), but the addition of wild-type PB1 would be expected to at least partially compensate for this. Thus this system allowed us to examine viral RNA production from the mutant segments in isolation from potentially confounding issues of segment delivery and PB1 protein function. Forty-eight hours post-transfection, RNA was harvested and primer extension analysis for segment 2 v, m and cRNA was performed. Omitting PB2 from the transfections determined the baseline levels of segment 2 vRNA that were expressed from the pPolI promoter ( Figure 7C , lane 1). In the presence of the full 3PNP complex, the mutant segment 2 constructs were transcribed and replicated to broadly similar extents ( Figure 7C ). When replicate experiments were quantified, the AUG1 mutants and the T32G AUG2 mutant accumulated vRNA to >75% of the WT level, in clear contrast to the >10-fold reductions they exhibited in the context of virus infection ( Figure 7B) . Similarly, all mutants expressed mand cRNA to reasonable levels, with, on average, no change of >2-fold compared to the WT ( Figure 7D ). These data argue against a defect in the promoter sequence of the viral RNA being solely responsible for the reduced levels of viral RNA seen in the context of infection and support instead the hypothesis that mutations around AUG1 not only affect translation initiation of PB1 and downstream cistrons, but also affect genome packaging.