The sequences surrounding AUG1 are highly conserved, and conform to a moderately strong initiation consensus (Table 1) . Upstream residues were modified to each of the other possibilities at the crucial À3 position in the T22A, T22G and T22C mutants ( Figure 1B) , with the A/G changes but not the U!C alteration expected to result in increased ribosome recognition of the AUG (23,24) ( Figure 1C) . A further set of segment 2 AUG1 variants were produced on the background of the previous mutants in which the upstream residues at positions À1 and À2 were changed to match the canonical Kozak consensus (ACC, CCC, TCC mutants). The initiation context of AUG1 was also weakened by mutating the G at the +4 position to an A (G28A); this change also resulted in a non synonymous D2N change in the predicted PB1 translation product ( Figure 1B and C). Similar approaches were taken to probe the function of AUG2 and 3 in regulating translation. The context of these initiation codons was improved by mutating the +4 nt to G (T32G and C74G respectively, which caused V3G and A17G changes in the PB1 ORF, respectively). Additionally, AUGs 2 and 3 were individually destroyed (T30C and T72C respectively) without altering the PB1 amino acid sequence. Two further mutations were made to examine the importance of AUG3/sORF2. A78T removes the stop codon from sORF2, and thus the predicted frame 2 protein product from this construct is an N-terminal fusion to PB1-F2. To investigate if the length and position of sORF2 was important (as in the case of a reinitiation event), a stop codon was re-introduced prior to AUG4, in the A78T+G101T mutant. This mutant left only 15 nt between the stop codon and AUG4, and also produced a G26V alteration in the PB1 sequence. Finally, AUG4 was removed by a T120C alteration, as used by many previous studies to ablate PB1-F2 expression (6, 10, 13, 14, 16, 17, 33, 34) . The positions of all mutations are shown on the PR8 segment 2 sequence in Figure and their predicted effects on AUG context and ORF structure are summarized in diagrammatic form in Figure 1C .