Structure probing analysis of the MNV signal revealed that, like the BM2 signal, the mRNA in the region essential for terminationreinitiation is not highly structured. This was especially evident from the chemical probes, with most residues sensitive to imidazole and lead cleavage. The enzymatic probes were also active against the majority of bases in the region and consistent with this, CV1 probing identified very few double-stranded or stacked bases. We also noticed a few CL3 cuts at residues other than C, although the reason for this is uncertain. Minimal free energy mfold predictions, performed using the online server of Zuker (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) indi- cated that the most stable RNA fold was the bulged stem-loop shown in Figure 4 . However, the correspondence between this mfold and the mapping data was not absolute. Whilst in general, the single-stranded probes displayed more activity against regions of the model predicted to be single-stranded than they did against predicted helices, there were anomalies. For example, residues G51-52 were sensitive to RNase T1, yet were predicted to be in a double-stranded region (stem 2). Generally, the predicted duplexes showed more reactivity to single-stranded probes than one would expect for stable double-stranded stretches. Therefore, it seems likely that the RNA in this region is metastable, potentially adopting a number of co-existing structures. In our model, the sequence complementary to 18S rRNA is sequestered between two putative stems (stems 2 and 3; Figure 4 ) at a location similar to that found with BM2 [19] . Given that the termination-reinitiation process requires the ribosome to translate through the VP1 ORF, secondary structure in the RNA upstream of the ''stop-start'' window would be unwound and perhaps remodelled as the ribosome transits to the termination codon. Toeprinting of ribosomes paused at initiation codons has shown that the 59 edge Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH-) and the position of known RNase U2 and T1 cuts, determined empirically. Products were analysed on a 10% acrylamide/7M urea gel containing formamide. Data was also collected from 6% and 15% gels (gels not shown). Enzymatic structure probing was with RNases T1, U2, CL3 and CV1. Uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). The number of units of enzyme added to each reaction is indicated. Chemical structure probing was with imidazole (I, hours) or lead acetate (Pb; mM concentration in reaction). The water lane (W) represents RNA which was dissolved in water, incubated for four hours and processed in parallel to the imidazole-treated sample. The sequence of the probed RNA and the inferred secondary structure is shown in Figure 4 . of the ribosome is some 12 to 13 nt from the first base of the AUG [21] . This would place the 59 edge of the terminating ribosome (with the UAA codon in the A-site) close to residue C78 on our mRNA. Thus a terminating ribosome would prevent formation of the secondary structure, conceivably releasing the 18S rRNA complementary region for interaction with the ribosome (see Discussion). An alternative structure can be predicted under such circumstances, shown in the inset box in Figure 4 . In this structure, the 59 arm of the original stem 2 is predicted to pair with alternative bases to generate a new stem (stem 29) with Motif 1 forming part of the apical loop. This alternative fold is attractive for a number of reasons. By displaying Motif 1 on an apical loop, this could promote 18S rRNA binding and ribosome tethering [19] . Until ribosomes transit through this region, Motif 1 would remain within a larger structure with potentially reduced access to the ribosome which could, at least in part, account for the observation that the signal does not appear to function as an IRES. The deletion analysis of Figure 1 is also consistent with a role for this alternative structure as the functional ''end-point'' maps to the start of the 59 arm of stem 29. Furthermore, most, if not all, viral TURBS have the potential for base-pairing between regions flanking Motif 1 [18] . Nevertheless, it should be noted that the structure mapping data are not fully consistent with this alternative structure, for example, there is considerable sensitivity to RNase T1 cleavage within the 59 arm of stem 29. This is considered further in the Discussion section.