Packaging signals have been previously mapped to the general area of the 3 0 -end of segment 2 vRNA (38-41) (summarized in Figure 9 ) but this is the first study to show that the same nucleotides also contribute to translational regulatory sequences. This finding echoes our previous finding that sequences important for directing packaging of segment 7 overlap other cis-acting signals for mRNA splicing (30) and further demonstrates the functional complexities contained within sections of the influenza A virus genome. Examining the sequence of the 5 0 -end of segment 2 (in mRNA sense) using the criterion of reporting sequences that are conserved in >95% of the available isolates makes it evident that the primary selection pressure acting on the region is PB1 function (50) . By this admittedly simple measure, only two amino acid residues (positions 12 and 14) are not conserved, in obvious contrast to PB1-F2 or sORF1 and sORF2. At the nucleotide level, as previously noted (50) , it is clear that the majority of sequence polymorphisms are found at the third base position of the PB1 gene ( Figure 9 ). Consistent with this, experimental evidence shows that the majority of the 14 N-terminal amino acids as well as (where tested) Figure 8 . Model for translation of segment 2 polypeptides. PB1 translation occurs by canonical initiation at the first AUG. The majority of PB1-F2 translation occurs via leaky scanning to bypass AUGs 1-3. In contrast, reinitiation after termination at the end of sORF2 is a major contributor to PB1-N40 translation. See text for further details. residues further downstream, are important for one or more functions of PA binding, polymerase activity and virus replication (21, (51) (52) (53) (and data presented in this study). However, although over half of the first 41 codons of PB1 show some variability at the wobble position, it is notable that the primary translational signals within this region are much more highly conserved, with all five start codons showing >99% conservation and only one of the two stop codons (that of sORF1) showing apparent variation (Figure 9 , Tables 1 and 2 ). Even here, as discussed, the variation is such that >99.9% of viruses maintain either a UGA or UAA stop codon (Table 2) .