Picornavirus mRNAs are translated by an internal initiation mechanism, in which the ribosome enters directly at an internal site within the mRNA rather than scanning from the physical 5' end. So far, internal ribosome entry sites (IRESs) have been identified for all the picornaviruses as well as an increasing number of cellular mRNAs. The IRES elements in picornaviruses have been located within the 5' non-coding region, where the sequence and the RNA secondary structure are well conserved among viral serotypes in each genera. Based on sequence alignments, the genome of DHV-1 showed a typical picornavirus genetic organization and a putative class IV IRES element within DHV-1 5'-UTR. Therefore, DHV-1 should use similar picornavirus-like strategies for the initiation of translation. In this paper, we demonstrate, for the first time, that the DHV-1 5'-UTR has typical IRES activity, and that it can drive the translation of a downstream reporter gene both in vitro and in vivo. According to the prediction of the IRES structure, the DHV-1 IRES belongs to the type IV HCV-like IRESes, but it shares some common features with the IRES of viruses included in different groups.
Some picornaviruses, such as the AEV, contain HCV-like IRES elements organized in two major secondary structure domains, II and III [35, 36, 8], in which all the structural elements crucial for initiation of translation are located. Mutations within the putative loop of domain IIIe and stem1, 2 inhibited the DHV-1 IRES function. Moreover, the restoration of these structures partially recovered the activity of the DHV-1 IRES. These data provide strong evidence for the presence of the conserved domains II and III, common to other picornaviruses, within the DHV-1 5'-UTR and for the relevance of these regions to keep the normal internal initiation of translation of the DHV-1.
We have demonstrated that the sequences included within the DHV-1 Vp0 coding region contribute to the efficiency of internal initiation. The relevance of the coding sequences located immediately downstream of the IRESes has been previously described for other RNA viruses, such as the HCV or CSFV [22, 24]. While most IRESs require only the 5'-non-translated region (5'-NTR) for full activity, the HCV or CSFV IRESes depend on the presence of protein-coding sequences downstream of the initiating AUG [37–39]. The minimal coding region elements required for high activity were exchanged between HCV and CSFV and the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the IRESes and the coding sequences . Although nucleotide sequence of this region is different in CSFV and in HCV, they share common features such as being A rich and not highly structured. In fact, the efficiency of internal initiation of these chimeric constructs correlated with the degree of single-strandedness of the region around the initiation codon. According to secondary structure prediction, the DHV-1 coding region located immediately downstream of the IRES is not highly structured. This sensitivity to secondary structure around the initiation codon could be due to the fact that the eIF4F complex (which includes the eIF4A helicase as one of its subunits) is not required for translation initiation on these IRESs. However, the interaction of cellular proteins with the coding regions adjacent to the IRESes has been proposed as an alternative mechanisms that enhance viral translation, as it is the case of the NS1-associated protein 1 (NSAP1) and the HCV IRES , and cannot be discarded in the case of DHV-1.
Finally, we have characterized the DHV-1 IRES requirement for the eIF4F complex components and, as well as the EMCV and HCV-like IRESes, is insensitive to the FMDV L protease and the 4E-BP1 activities. These results indicate that the translation initiation takes places in the absence of fully functional eIF4E and eIF4G proteins. However, while the DHV-1 and HCV-like IRESes are not inhibited by hippuristanol or dominant negative forms of eIF4A, the EMCV IRES is strictly dependent on the eIF4A function . Although the EMCV and DHV-1 IRESes share some common structural organization, they initiate translation by different mechanisms involving different cellular translation initiation factors. Moreover, as the DHV-1 and HCV share common structural motifs, they have similar requirement for the eIF4F complex components. Thus, it is likely that the translation initiation of DHV-1 could proceed similarly to PTV-1 and to other HCV-like IRES containing picornaviruses, by the direct recruitment of the 40S ribosomal subunit to the viral mRNA.
All together, these results demonstrate, for the first time, the existence of an IRES element within the highly structured 5'-UTR region of the DHV-1 genome. The DHV-1 IRES could be classified as type IV IRES attending to its secondary structure and biological functions and, consequently, it exhibits functional similarities with the HCV IRES. These new advances in the understanding of the DHV-1 IRES structure, function and interaction with translation-initiation factors provides a foundation for developing therapeutics to prevent the viral protein synthesis.