Segment 2 mRNA is known to encode three polypeptides: PB1 and PB1-N40 in frame 1, and PB1-F2 in frame 2 (6,7), translated from AUGs 1, 5 and 4 respectively ( Figure 1A ). The sub-optimal Kozak consensus flanking AUG1 prompted the hypothesis that the PB1-F2 ORF is accessed by leaky ribosomal scanning (6) and consistent with this, we previously showed that there was increased translation from AUG5 in the absence of AUG4 (7). However, there are two intervening start codons in frame 2 between AUG1 and the PB1-F2 ORF that initiate short sORFs with minimal protein coding capacity; eight and two codons respectively ( Figure 1A and B). Nevertheless, both these AUGs are highly conserved, being present in >99% of the available segment 2 sequences, similar to the conservation shown by AUGs 4 and 5 for PB1-F2 and N40 respectively (Table 1) . Notably, the termination codons for these sORFs are also highly conserved in that although three of the four overlapping PB1 codons in frame 1 are not themselves highly conserved at the RNA level, a stop codon is almost always (>99.9% for both sORF1 and 2) maintained in frame 2 ( Table 2 ). This degree of conservation is suggestive of functional importance, potentially for the regulation of translation of the downstream PB1-F2 and N40 cistrons. Accordingly, we set out to further delineate the mechanisms controlling translation from segment 2 mRNA by systematically introducing mutations into AUGs 1-4 and their flanking regions that would be predicted to alter their usage.