Abstract
Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. The genomes of these viruses contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The activity of the RhPV 5' IRES element has previously been demonstrated in mammalian, insect and plant translation systems. It has also been shown that the RhPV 5' IRES forms 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and elFl. It is also possible to delete large regions of the 5' IRES without affecting initiation complex formation. We have now defined the minimal sequences required for directing internal initiation in mammalian, plant and insect translation systems. Fragments of around 130 nt from the 3' portion of the 5'untranslated region (UTR) can direct translation in each of these systems. Thus, the 3' region within the 5'UTR seems to be critical for IRES function. Interestingly, this region is mainly unstructured, making the RhPV 5'IRES unique among viral IRES elements. The RhPV IRES was also compared to the 5'UTR of the insect iflavirus Kakugo virus (KV). Iflaviruses had been initially classified as insect picoma-like viruses alongside the Dicistroviridae. The KV 5'UTR was shown to contain an IRES element similar in function to that of RhPV. Since IRES elements have been shown to require trans-acting factors (ITAFs) for their activity, in addition to the canonical initiation factors, we investigated the interactions of the RhPV IRES with cellular proteins of mammalian, plant and insect origin. In our preliminary studies we identified proteins with a potential role as ITAFs. The potential advantages of the RhPV IRES in biotechnology were assessed in conjunction with a Sindbis virus-based expression system. Like the RhPV 5'IRES, this system is able to function in mammalian and insect systems. However, our preliminary experiments in mammalian cells were not able to clearly establish the advantages of the RhPV 5'IRES in such a system.