The translation of in vitro synthesised wild-type (wt) mRNA from p2luc-MNVwt was carried out in FlexiH rabbit reticulocyte lysate (FlexiHRRL) supplemented with 140 mM KCl (see Materials and Methods) and gave products of the expected sizes (upstream rlucVP1 ORF, ,42 kDa, downstream VP2fluc ORF, ,64 kDa, Figure 1b ). The molar ratio of VP2fluc to rlucVP1 (taking into account the methionine content of the two proteins) was typically in the region of 1:10. Thus, initiation on the downstream ORF occurred at a frequency of about 10% of that of the upstream ORF. That this was indeed the product of the second ORF was further confirmed by comparing the migration of RRL translation products from mRNAs derived from p2luc-MNVwt that had been linearised at different points within the second ORF (data not shown). Termination-reinitiation is distinct from IRES-mediated expression of downstream ORFs as translation through the upstream ORF is an absolute requirement [11, 14, 16] . In order to establish whether this is also the case for MNV expression, a premature in-frame stop-codon was inserted close to the end of the rluc ORF but upstream of VP1 sequence information (219 bp upstream of the authentic rlucVP1 termination codon). If the expression of VP2fluc is a result of termination-reinitiation, translating ribosomes would be unable to reach the AUG start Figure 1 . Minimal sequence requirements for MNV termination-reinitiation. A) Schematic of the p2luc-MNV reporter mRNA. The termination-reinitiation region (203 nt upstream and 52 nt downstream of the UAAUG motif) was cloned into the SalI and BamHI sites of the p2luc reporter plasmid. HpaI run-off transcripts for in vitro translation were generated using T7 RNA polymerase. The location of the T3 promoter present in the structure mapping construct p2luc-MNV-T3 is indicated. B) Deletion analysis of MNV termination-reinitiation. A series of p2luc-MNV variants were prepared with stepwise, in-frame deletions from the 59 end of the inserted viral sequence. The wild-type (wt), premature stop (ps) and deletion mutant plasmids were linearised with HpaI and run-off transcripts translated in FlexiH RRL at a final RNA concentration of 50 mg/ml in the presence of [ 35 S]-methionine and 140 mM added KCl. The products were resolved by 12% SDS-PAGE and visualised by autoradiography. The number of nucleotides of viral sequence remaining up to the AUG start codon of the MNV ORF is shown below the gel. The product of the full-length or truncated versions of the rlucVP1 ORF (predicted size of MNVwt is 42 kDa) is marked rluc, and the VP2fluc product (predicted size, 62 kDa) is marked fluc. The MNV ps rluc product is the shortest (predicted size, 33 kDa). RRF denotes the relative reinitiation frequency in comparison to MNVwt (set at 100). The figure in brackets represents the ratio of the intensity of the fluc and rluc products (adjusted for methionine content and expressed as a percentage) for the MNVwt mRNA. doi:10.1371/journal.pone.0008390.g001 codon of VP2fluc in the mutant mRNA and the ORF could not be translated. As is clear in Figure 1b , the introduction of a premature stop codon into the rluc/M1 ORF abolished expression of the VP2/fluc product, but had no effect on synthesis of the upstream ORF (rlucVP1ps, ,33 kDa). These data are thus consistent with a termination-reinitiation strategy for the expression of the VP2 protein and confirm a requirement for translation through the upstream ORF.