Low level expression of the replicative machinery is a general feature of many viruses and in some cases, mutations that result in overexpression of the viral polymerase have been shown to be deleterious to virus fitness (36, 37) . It was therefore possible that the poor replication of the ACC, CCC and TCC mutants resulted from overexpression of PB1, although as noted above, the T22A, T22C and T22G viruses did not show growth defects. More generally, we wished to compare segment 2 protein expression from recombinant and authentic viral settings. Accordingly, PB1, N40 and PB1-F2 accumulation in MDCK cells infected with the panel of viruses was examined at 8 h post-infection (p.i.) by western blotting. To provide numerical data, the levels of each protein from replicate experiments were quantified and normalized to levels in cells infected with WT PR8. The results obtained could be divided into those that were in concordance the previous in vitro analysis, and those that differed. In agreement, weakening the Kozak consensus of AUG1 (G28A) reduced PB1 accumulation relative to N40 and F2 expression ( Figure 4A , compare lanes 2 and 9; quantification in Figure 4B ). Similarly, mutation of AUG2 had little effect on either the quantity (T30C) or the relative ratios (T32G) of the segment 2 polypeptides ( Figure 4A, lanes 12 and 13) , with the lower levels of all three polypeptides seen with the latter virus being plausibly ascribed to the reduced polymerase activity of the mutant PB1 protein, as NP accumulation was also reduced. Also in agreement, loss of AUG4 increased N40 synthesis by $6-fold (T120C; lane 20), as we previously observed for a similar mutant virus (7).