If PB1-F2 is accessed by leaky scanning but N40 is accessed by reinitiation after translation of sORF2 then the insertion of further AUG codons in the region between the end of sORF2 and the beginning of the PB1-F2 ORF at AUG4 would be predicted to decrease F2 expression (through 'soaking up' initiation competent scanning ribosomes) but to have little effect on N40 expression because they would be effectively invisible to scanning small subunits that had terminated after reading sORF2 but not yet had time to acquire new initiation factors. To test this hypothesis, we introduced mutations that created new strong context AUG codons in each of the three reading frames in this region ( Figure 5A ). To permit the analysis of mutations that were lethal to virus growth and to try and minimize the effects of differing protein stability on polypeptide accumulation, we created sets of chimaeric plasmids containing the 5 0 -end of segment 2 encompassing the PB1-F2 coding sequence followed by a GFP ORF such that either PB1 (frame 1) or PB1-F2 (frame 2) were fused in frame ( Figure 5B ). To validate this system, we first retested the effect of the key mutations affecting sORF2 and AUG4. Cells transfected with a plasmid encoding the WT segment 2 fused in frame 1 to GFP produced the expected ratio of PB1 and PB1-N40-derived fusion proteins, while the frame 2 fusion Figure 5D ). Similarly, fusion of sORF2 and the F2 ORF by the A78T mutation significantly reduced N40 production ( Figure 5C , compare lanes 10 and 11). In contrast to virus infection, this mutation also increased accumulation of the F2-fusion polypeptides, presumably because of the greater stability conferred by the GFP moiety. Both the decrease in N40 expression and the increase in F2 synthesis were reversed by reinstating the sORF2 stop codon by the further mutation G101T (lane 12). Thus this system successfully recapitulated the regulatory effects seen in the context of authentic virus infection.