Efficient termination-reinitiation seems to require the close proximity of the stop and start codons [12, 14, 19] . To investigate whether this is also the case for MNV, the authentic stop codon of rlucVP1 (in the context of the fully functional MNV49; see Figure 1b ) was mutated from UAA to CAA such that the first ORF was extended by 13 amino acids (MNV49.1 Figure 5 ). The separation of stop and start codons by such a distance in BM2 is known to reduce reinitiation about 10-fold [19] , but with the MNV signal, only a three-fold reduction in flucVP2 synthesis was observed ( Figure 6 ). A possible explanation for this lies in the fact that the ''new'' stop codon is itself embedded within a second potential stop-start sequence (UGAUG) which could facilitate some reinitiation, but perhaps at a lower frequency, as it would not necessarily be spaced appropriately with respect to Motif 1. Another possibility is that 40S subunits terminating at the downstream stop-start sequence can reinitiate, at a reduced frequency, at the correct (upstream) AUG, despite the increased spacing, with the 40S subunit remaining tethered to the mRNA and ''snapping-back'' to the normal position of reinitiation. In an attempt to distinguish between these possibilities, additional constructs were prepared in which point mutations were introduced into pMNV.49 such that the termination and start codons in the two stop-start regions were changed separately and in combination (MNV49.2 to MNV49.8; see Figures 5 and 6) . From this analysis, it is evident that modification of the authentic termination-reinitiation motif reduces reinitiation, irrespective of whether the stop or start codon is eliminated. Alteration of the AUG codon had the most effect, with reinitiation reduced to 8-38% of the wild-type level. When the natural termination site was changed such that termination now took place 13 or 15 amino acids downstream, the frequency of termination-reinitiation was also reduced, to 25-49% of the wild-type level. In MNV49.8, where termination of the upstream ORF occurred 30 amino acids downstream of the natural site, very little reinitiation was seen (8% of the wild-type level), indicating that the ribosome is unable to locate the authentic AUG from such a distal termination site. In the translation of this mRNA, an additional product was seen (asterisked in Figure 6 ) whose size is consistent with a fusion of the encoded ORFs (this is considered in the Discussion section). Reinitiation events that take place at either the authentic or the downstream stop-start motifs would produce polypeptides that differ in size by only 13 amino acids, thus we would not expect to be able to distinguish them by SDS-PAGE, and this is clear in Figure 6 , where the reinitiation products show very similar electrophoretic mobilities. Thus we cannot say with confidence whether a particular AUG (or both) is used. However, the substantial reintiation activity displayed by MNV49.7, an mRNA in which both AUGs were changed, indicates that non-AUG codons can act as reinitiation codons, although probably at reduced efficiency. This is consistent with other work demonstrating that reinitiation can occur at non-AUG codons within the context of a termination-reinitiation signal [12, 14, 19] . Whilst in principle, reinitiation of translation of the MNV 49.7 VP2fluc ORF, following termination, could occur at the next available AUG, this is located 54 amino acids from the natural stop-start signal and initiation here would produce a substantially shorter product that would have been detectable by SDS-PAGE. Thus in this mRNA, a significant proportion of ribosomes (25% of the wild-type level) that terminate 13 amino acids downstream of the authentic stop-start site can reinitiate in an AUG-independent manner within the stop-start window.