Our data further refine the model for segment 2 protein expression. PB1 translation occurs via the canonical pathway of eukaryotic translation initiation (22) in which a preinitiation complex consisting of an eIF2aternary complex (eIF2-TC) attached to a 40S ribosomal subunit scans 3 0 -wards from the 5 0 -cap structure, recognizes AUG1 and commences translation after loss of the initiation factors and recruitment of the 60S subunit ( Figure 8A ). The simplest explanation for PB1-F2 expression is that it occurs via leaky ribosomal scanning, in which the preinitiation complex misses the moderate context AUGs 1 and 3 and the poor context AUG2 before initiating translation at the strong context AUG4 ( Figure 8B ). AUG3/sORF2 evidently plays an important role in down-regulating use of AUG4, as its loss through the T72 mutation substantially increased PB1-F2 accumulation, in vitro and in the context of virus infection. In contrast, the presence of AUG3/sORF2 up-regulated N40 expression in infected cells, a finding inconsistent with leaky scanning. Instead, we think this is best explained via leaky ribosomal scanning to bypass AUGs 1 and 2 followed by initiation at AUG3, almost immediate termination at the end of the two codon sORF2 and continued scanning of the 40S ribosomal subunit. The 40S subunit then scans past the strong context AUG4 but has time to reacquire an eIF2-TC before reaching the strong context AUG5 whereupon translation initiation occurs ( Figure 8C ). The distances between the sORF2 stop codon and AUGs 4 and 5 (40 and 63 nt respectively) are consistent with previously characterized instances of reinitiation (22, (45) (46) (47) . In some circumstances, changes in levels of eIF2-TC during conditions of cell stress (as for example when virus infection activates PKR) are known to regulate expression of downstream ORFs accessed via reinitiation strategies (22) . It is therefore interesting to speculate that segment 2 translation might be further regulated during the course of infection. The distance between the extended sORF2 in the A78T mutant and the N40 AUG (39 nt) is similar to that between the normal sORF2 and the PB1-F2 AUG, so we would not rule out the possibility that reinitiation after sORF2 translation also contributes to F2 expression. However, shortening the intercistronic distance between sORF2 and the F2 ORF to 18 nt (a distance predicted to be too short to allow efficient reacquisition of an eIF2-TC) in the A78T+G101T mutant did not significantly reduce F2 accumulation, so we do not think it plays a major role.