To investigate the reinitiation hypothesis further, the AUG3/sORF2 mutations were made on a ÁAUG4 (T120C) virus background ( Figure 4C ). These viruses were rescued and grew to comparable titre to wild-type PR8 (data not shown). Western blotting was performed on cells infected with these viruses, as well as (for comparison) from cells infected with the 'parental' T72C, C74G, A78T and T120C. As before, preventing potential reinitiation from AUG3/sORF2, using either the T72C or A78T mutations in the presence of an intact AUG4 reduced levels of N40 ( Figure 4A , compare lanes 19, 21 and 25), while improving the predicted strength of AUG3 had little effect (lane 23). Also as before, removal of AUG4 (T120C) led to a large increase in N40 levels (lane 20). A very similar outcome was obtained when the Kozak consensus of AUG3 was improved by the C74G mutation (C74G+T120C; lane 24). When AUG3 and 4 were removed concurrently (T72+T120C), synthesis of N40 was increased even further, to $14-fold greater levels than with the WT virus ( Figure 4B lane 22, quantification in Figure 4B ). However, when sORF2 was fused to the F2 ORF in the absence of AUG4, only a small enhancement (on average, 1.4-fold over WT) of N40 synthesis resulted (lane 26). If N40 expression was purely dependent on leaky scanning to bypass AUGs 3 and 4, this combination of mutations should have behaved identically to T120C alone. Instead, the absence of AUG4 only makes a significant difference to N40 expression when either sORF2 terminates before AUG5 or AUG3 is also absent, consistent with a reinitiation mechanism for synthesis of N40.