The in vitro translation of the reporter proteins was evaluated measuring 35S-methionine incorporation. Cap-dependent translation from the first ORF was determined by the level of CAT expression and internal initiation activity of the different sequences included between the two reporter genes was estimated by the accumulation of fLUC. All the mRNAs expressed CAT efficiently as expected. Moreover, plasmids containing the EMCV IRES and the DHV-1 5′-UTR sequence in sense orientation allowed the efficient expression of fLUC. These results suggest that the DHV-1 5′-UTR sequence is able to efficiently initiate cap-independent protein synthesis in vitro.
To confirm and extend the results from these in vitro assays, the same dicistronic plasmids were tested in vivo by transient-expression experiments in mammalian cells. The dicistronic plasmids were transfected into vFT7-infected BHK-21 cells, and 20 h post-transfection, cell extracts were prepared and protein synthesis was analyzed by SDS-PAGE and immunoblotting to detect CAT and LUC expression. As expected, all plasmids expressed CAT efficiently. Moreover, the DHV-1 5′-UTR and the EMCV IRES containing plasmids also produced efficient fLUC accumulation. These results indicated that the putative DHV-1 IRES element is able to efficiently initiate protein synthesis in vivo.
Stem1, Stem2 and domain IIIe are essential to the function of DHV-1 IRES
Although there is only limited primary sequence relatedness among the 5′-UTRs of DHV-1, AEV and HCV, the structure of domain IIIe appears remarkably conserved among these viral IRESes [2,35,11]. It has been previously reported that domain IIIe is critical for the HCV, PTV-1 or AEV IRES-mediated initiation of translation. To evaluate the relevance of the putative IIIe region in DHV-1 IRES activity, the most important loop sequence within the IIIe region (GAUA) was mutated to AAAA (DHV IIIe mut) (Figure 3A) and the effect of the mutation on the translation initiation was evaluated. Mutant constructs were analyzed both in Flexi RRL and in DF-1 cells. The results reveal that these mutations partially inhibited translation initiation both in vitro and in vivo, confirming the relevance of domain IIIe for DHV-1 IRES activity. Surprisingly, the reduction in the translational activity of the DHV-1 IRES upon disruption of the domain IIIe (50%) is moderate when compared with similar mutations in other type IV IRESes (90-95%).
Different experiments carried out with other picornaviruses, such as the HCV, PTV or AEV, showed that stem 1 and stem 2 within the IRES structure are crucial for translation initiation. The disruption of the base pair interactions in these regions seriously damages the ability to initiate translation. To analyze the role of the putative stems loops of the DHV-1 IRES, different mutations were introduced to disrupt the predicted base pairing of stem 1 (DHV stem1 mut) and stem 2 (DHV stem2 mut). The mutant constructs were analyzed by triplicate both in Flexi RRL and in DF-1 cells. The results showed that both mutations resulted in a slight reduction in translation initiation ability of DHV-1 IRES both in vitro and in vivo. Moreover, the function of the IRES was partially recovered with the corresponding mutations that restore the structure (DHV stem1 mut’ and DHV stem2 mut’ respectively), confirming that the predicted pseudo-knot structures are formed in vivo and that they play a role in viral translation regulation. Once more, the reduction in the translational activity of the DHV-1 IRES upon disruption of these structures (50%) is moderate when compared with similar mutations in other type IV IRESes (90-95%).