Initially, PB1 and N40 protein synthesis by the mutants was investigated using coupled in vitro transcription and translation (IVT) in rabbit reticulocyte lysate. As expected (7) wild-type (WT) PR8 segment 2 expressed both PB1 and N40, with preferential usage of AUG1 ( Figure 2A , lane 1). All changes tested in the À3 to À1 positions relative to AUG1 lead to notable increases in the expression of PB1 (lanes 2-7). Quantification of replicate experiments showed these increases to be at least 2-fold relative to the WT gene ( Figure 2D ). Although this was the predicted outcome when the U at the À3 position was swapped to a purine, a similar effect when it was replaced with another pyrimidine was unexpected (23) . Consistent with a role for leaky scanning in accessing downstream AUGs, a concomitant reduction in N40 levels was observed in all cases ( Figure 2D ). Conversely, weakening the Kozak consensus of AUG1 through replacement of the +4 G nucleotide downregulated PB1 expression, and although the absolute amount of N40 remained similar ( Figure 2A , lane 8, quantification in Figure 2D ), its ratio relative to PB1 was increased 1.7-fold compared to wild-type segment 2. In contrast, mutations affecting AUGs 2 or 3, whether by altering their context (T32G and C74G) or by destroying them (T30C and T72C) had little effect on the ratio of PB1 to N40 ( Figure 2B ; quantification in Figure 2D ). Similarly, increasing the length of sORF2 (A78T+G101T) or fusing it with the PB1-F2 cistron (A78T) did not substantially alter relative use of AUGs 1 and 5. However, loss of the PB1-F2 AUG4 through the T120C mutation increased N40 synthesis from AUG5 by nearly 3-fold ( Figure 2B , lane 8, Figure 2D ). This recapitulated our previous observation for a construct in which AUG4 was mutated and its surrounding Kozak consensus disrupted [ÁAUG; (7)]. The small size ($10 kDa) of the PB1-F2 polypeptide made it difficult to visualize directly in IVT reactions. Accordingly, we utilized a set of constructs in which the CAT gene was fused downstream of AUG5 to increase the size of the polypeptide products. Examination of IVT reactions programmed with plasmids containing the CAT gene inserted into WT segment 2 in each of the three reading frames showed the expected set of polypeptides: frame one produced PB1 and PB1-N40 fusion proteins, frame 2 produced a PB1-F2 fusion while frame 3 lacked an obvious polypeptide product ( Figure 2C , lanes 1-3 respectively). The quantity of the frame 2 product synthesized showed partial correlation with the presence and number of upstream AUG codons. For example, improving the Kozak consensus of AUG1 had only a small effect on PB1-F2-CAT synthesis (T22A: Figure 2C , lane 4; quantification in Figure 2D ) and nor was F2 synthesis increased by the G28A mutation that weakened the context of AUG1 ( Figure 2C, lane 5) . Similarly, alteration of AUG2 by the T30C and T32G mutations had little effect (lanes 6 and 7). In contrast, the level of PB1-F2 was up-regulated 2-fold when AUG3 was removed via the T72C change (lane 8). The C74G mutation, predicted to improve the Kozak consensus of AUG3 caused a slight (<2-fold) reduction in F2 expression that was not statistically significant (lane 9). These results suggested that while AUG2 was of little translational significance, AUG3 was recognized well by ribosomes and directly regulated the level of PB1-F2 expression. Supporting this, when sORF2 was fused to the F2 ORF through removal of the intervening stop codon in the +2 frame by the A78T mutation, increased levels of a slightly longer (presumed) fusion protein were seen (lane 10). Thus overall, the data suggested that expression of PB1-F2 and PB1-N40 in vitro could be partially but not wholly explained by leaky ribosomal scanning. AUGs 1, 3 and 4 were clearly functional and exerted a significant influence on use of downstream start codons. The poor context AUG2 however, was apparently not used.