Next, we tested the effect of introducing novel AUG codons between the termination codon of sORF2 and AUG4. Insertion of new AUG 'A' into the PB1 ORF by mutating glycine codon at position 26 to ATG resulted in the production of a prominent novel frame 1 product ('PB1-N26') as well as a significant reduction in the synthesis of the PB1-F2 fusion protein ( Figure 5C , compare lanes 2 and 5, quantification data in 5D). N40 synthesis was however unaffected. These effects were specific to the creation of a new AUG codon, since mutation of codon G26 to ATC left expression of PB1-F2 unaltered (lane 6). Similarly, when new AUG codon 'B' was introduced into frame 2 by mutation of PB1 codon T25 to TAT (with the 5 0 -A!T change avoiding the simultaneous introduction of a stop codon in frame 3; Figure 5A ), N40 expression was not significantly altered while F2 accumulation was substantially reduced, partially at the expense of a slightly longer N-terminally extended form (lane 7). Again, the effect was specific to the AUG codon rather than mutation of codon 25 per se, because its mutation to CGT (B ctr) left F2 expression unchanged. N40 expression was also insensitive to the introduction of an AUG codon into frame 3, whereas F2 accumulation was reduced >2-fold (codon 'C'; lane 13). Once again, the paired control mutation had no affect on PB1-F2 synthesis, although unexpectedly, this change increased N40 accumulation (lane 14). Overall therefore, PB1-F2 levels were sensitive to the presence of start codons in all three frames following sORF2, whereas PB1-N40 levels were not significantly affected. These data show a fundamental difference in how AUG codons 4 and 5 are accessed: ribosomes can be diverted away from AUG4 by the insertion of new upstream AUG codons in the 'UTR' following sORF2, but AUG5 is insensitive to this approach. The simplest explanation consistent with the data is that AUG4 is primarily accessed by leaky ribosomal scanning that bypasses AUGs 1-3, while AUG5 is reached by reinitiation of ribosomes that have recently terminated synthesis after translation of sORF2.