The other major source of divergence between the in vitro data and that observed from the virus infections was seen with the mutants where the Kozak consensus of AUG1 was up-regulated. While the levels of N40 and PB1-F2 were predictably reduced, in all cases, the cells infected with these mutants also underexpressed PB1 relative to the WT virus ( Figure 4A, lanes 2-8) , showing on average 20-80% reductions in PB1 accumulation ( Figure  4B ). This was most pronounced for the triple AUG1 mutants, ACC, CCC and TCC, which produced 18, 32 and 47% of the PB1 levels of WT virus respectively. This was in marked contrast to the in vitro translation data, where PB1 levels were increased $2-fold over WT ( Figure 2 ). However, it should be noted that when the ratios of the three segment 2 polypeptides were considered, their relative amounts changed as predicted for leaky ribosomal scanning: N40 to PB1 levels were reduced between 2-fold (T22G) and 1.5-fold (ACC) in the AUG1 up mutants while PB1-F2: PB1 ratios decreased on average by $3-fold. Furthermore, transfection experiments confirmed that the AUG1 up mutations produced elevated amounts of PB1 in a cellular environment when introduced via plasmid (data not shown). We therefore considered the alternative hypothesis that in the background of authentic viruses, the AUG1 mutations also perturbed segment specific packaging. It is well established that the terminal unique coding and non-coding regions of all segments (including the regions of segment 2 under investigation here) contain specific packaging signals (3, (38) (39) (40) (41) (42) . In this hypothesis, the growth defect of the AUG1 mutants and their failure to express normal, let alone elevated quantities of PB1 could be explained by reduced delivery of segment 2 to the infected cells because of underincorporation of the segment into virions.