An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators

Fig. 1.. Analysis of 2A and 2B* expression in V5-tagged wild-type (WT) and mutant EMCVs. (a) Map of the ~7700 nt EMCV genome. The 5′ and 3′ UTRs are indicated in black and the polyprotein ORF is indicated in pale blue with subdivisions showing mature cleavage products. The overlapping 2B* ORF is indicated in pale pink. (b) Mutations introduced to prevent PRF (SS), and StopGo-mediated co-translational separation at the C-terminus of 2A (LV). Additional mutants were constructed with sequence encoding a V5 tag. (c–e) Western blot analysis of virus-infected cell lysates. BHK-21 (c) or L929 (d, e) cells were infected with V5-tagged WT or mutant viruses at m.o.i.~10 and lysates prepared at 8 h post infecton (p.i.). Anti-V5 antibodies were from rabbit (c) or mouse (d, e) along with the appropriate secondary antibody labelled with IRDye 680. Antibodies Mu-anti-2A, anti-2B*(N-term) and anti-2B*(C-term) were used with the appropriate secondary antibody labelled with IRDye 800. Left and right panels show scans from, respectively, the 700 and 800 nm channel of images obtained with a LiCor Odyssey scanner. Different markers were used for (c) and (d, e) with some differences between the two. (f)…

Fig. 2.. EMCV frameshifting efficiencies measured by metabolic labelling. (a) Phosphorimager image of SDS-PAGE of lysates of L929 cells infected with WT or mutant viruses. Positions of EMCV proteins are indicated. (b) Ratio of band intensities between WT and SS mutant viruses. (c) Mean ratio of measurable polyprotein products encoded downstream of the frameshift site to products encoded upstream of the frameshift site, normalized by the SS mutant. Values in (b, c) show means±sd of three biological replicates.

Fig. 3.. Role of a 3′ stem-loop structure in frameshift stimulation. (a) Mutations introduced to destabilize the predicted stem-loop structure (SL5′, SL3′, SL5′a, SL3′a) or restore it with altered base-pairings (SL5′3′, SL5′3′a). Mutated nucleotides are indicated with pink boxes. Mutants were generated in the context of a parent virus, WT-PTC, in which the 2B* ORF was truncated via the introduction of two PTCs. NPGP to NPLV StopGo mutant versions of the six stem-loop mutants and the WT-PTC parent were also constructed. (b) Phosphorimager image of SDS-PAGE of lysates of L929 cells infected with WT or mutant viruses. The positions of EMCV proteins are shown on the right. Note that Fig. 2(a) is identical to the left-hand portion of this figure, reproduced here for clarity. (c) Mean ratio of measurable polyprotein products encoded downstream of the frameshift site to products encoded upstream of the frameshift site, normalized by SS mutant virus. The red dotted line at 1 indicates the SS mutant ‘no frameshift’ baseline. (d) Direct PRF efficiency estimates based on 2A-2B*(PTC) and 2A-2BN expression in the three LV PTC mutants with a quantifiable 2A-2B*(PTC) band. Values in (c, d) show means±sd…