Previous work has revealed that termination-dependent reinitiation on the BM2 ORF of segment 7 of influenza B virus is dependent on a relatively short (45 nt), largely unstructured, TURBS containing a typical motif 1 element towards the 59 end [11] . In the present study, we investigated features of the TURBS required for efficient reinitiation, focusing primarily on the proposed interaction between TURBS motif 1 and helix 26 of 18S rRNA. Whilst the effects of point mutations within motif 1 are consistent with such an interaction, direct evidence is lacking in the BM2 system. We began by attempting to block the interaction by targeting the binding partners with antisense oligonucleotides. Such an approach was successfully employed in studies of the IRES-like properties of the Gtx leader (which recruits ribosomes de novo by virtue of interactions between the 18S rRNA and the mRNA) and revealed that the IRES activity could be blocked by AONs that bind either the mRNA or the rRNA [36] . Whilst we were able to observe specific inhibitory effects with an oligonucleotide that targeted motif 1, no effect was observed with an oligonucleotide targeting the ribosomal RNA ( Figure 2 ). Our failure to observe an effect of the AON complementary to the apical loop of helix 26 may be due to a failure of the AON to access the target. The Gtx mRNA:rRNA interaction occurs at a In the p2luc-BM2Gtx mutants, the 7 nt core of motif 1 (bold italics in p2luc-BM2wt) was substituted for nucleotides complementary to the region of 18S rRNA where the Gtx IRES is believed to bind. Note the p2luc-BM2Gtx Rabbit sequence is altered from that of the p2luc-BM2Gtx Mouse reporter such that 100% complementarity with the different rabbit 18S rRNA could be achieved. Nucleotides differing between Gtx Mouse and Gtx Rabbit are underlined. RNAs were translated and analysed as in the legend to Figure 1 . A series of previously described motif 1 mutants [11] were also translated as controls. doi:10.1371/journal.pone.0016822.g003 different region of helix 26 than the putative segment 7 mRNA:rRNA interaction, and this may be more accessible. Indeed, termination-reinitiation could not be reconstituted in reporter mRNAs in which motif 1 was replaced by the Gtx 18S rRNA binding motif ( Figure 3C) , and thus different responses to oligonucleotides targeting the rRNA may be expected. It may also be that the 18S rRNA-targeting oligonucleotide may need to adopt a similar structure to the mRNA if it is to effectively bind to the 18S rRNA and block reinitiation. The inhibition of termination-reinitiation by the AON that targeted motif 1, in contrast, is highly consistent with its proposed role in binding to helix 26, although effects via RNA conformation or binding to an unknown molecule cannot be ruled out. However, the weight of evidence from mutational analysis, the yeast data described below and the AON titrations strongly suggest that the inhibitory effect of AONs directed against motif 1 are likely due to their blocking of the interaction between mRNA and 18S rRNA.