The 5′ Untranslated Region of Rhopalosiphum padi Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems

Abstract

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a singlestranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5 UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5 UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5 IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries. Rhopalosiphum padi virus (RhPV) is an insect virus with a narrow host range, infecting aphids of the Rhopalosiphum andSchizaphis families (6, 11). Virus infection reduces both the life span and the reproductive capacity of the insects (6). RhPV was initially classified within the Picornaviridae, based largely on its physicochemical properties. However, sequence analysis has prompted a reevaluation of this attribution, and RhPV is now considered to belong to a group of insect viruses (the cricket paralysis-like viruses) with a picornavirus-like capsid structure but a distinct genome organization (15,16). Other members of this group include Drosophila C virus, Plautia stali intestinal virus (PSIV), and cricket paralysis virus (CrPV) itself. The RNA genome of each of these viruses, including RhPV, encodes two polyproteins in separate open reading frames (ORFs) (15). ORF1 encodes nonstructural proteins that possess sequence similarity to both mammalian picornavirus and plant comovirus proteins. ORF2 encodes the three structural proteins that also show similarity to picornavirus capsid proteins. Both ORFs are preceded by long untranslated regions (UTRs) about 500 nucleotides (nt) long. In contrast, mammalian picornaviruses encode one long polyprotein with the structural proteins at the N-terminal region and the nonstructural proteins at the C terminus. The genome organization of the cricket paralysis-like viruses resembles that of the caliciviruses (23). However, there is no evidence for the production of a subgenomic RNA by these insect viruses, and calicivirus ORFs are not preceded by long UTRs.It is well established that the initiation of translation on picornavirus RNA oc...

New insights into internal ribosome entry site elements relevant for viral gene expression

“…Thus, translation of each open reading frame depends on an active IRES element with functional and structural features that differ from any of the IRES elements described above. Indeed, the 59 UTR of Rhopalosiphum padi virus (RhPV) has been reported to contain an IRES that is active in plant and mammalian cellfree translation systems (Groppelli et al, 2007;Woolaway et al, 2001), and can assemble 48S initiation complexes in vitro with eIF2, eIF3 and eIF1 (Terenin et al, 2005) ( Table 1). Two separate unstructured regions within the 59 UTR seem to be critical for IRES function, but sequences of the 59 UTR can be deleted without affecting initiationcomplex formation.…”

A Cross-Kingdom Internal Ribosome Entry Site Reveals a Simplified Mode of Internal Ribosome Entry

“…1. The entire 5Ј-UTR of RhPV RNA linked to the coding region for the N-terminal portion of the luciferase reporter was used throughout these assays, as it was shown earlier that this sequence does not significantly alter the IRES activity compared to the native coding region (47). It should be noted that all RNAs used in these experiments contained a stable stem-loop structure at their 5Ј ends as an additional block to the 5Ј end-dependent translation initiation.…”

“…Recently, the 579-nucleotide-long 5Ј-UTR of Rhopalosiphum padi virus (RhPV) mRNA has been shown to contain an IRES that functions efficiently in mammalian, plant, and insect in vitro translation systems (38,47). This suggests that the RhPV IRES may employ a simplified mode of internal ribosome entry.…”

“…Unlike the intercistronic region, the 5Ј-UTR of RhPV RNA initiates translation at an AUG codon using the initiator tRNA. Strikingly, some large deletions in both the 3Ј-half and the 5Ј-half of the RhPV 5Ј-UTR do not abrogate the RhPV IRES activity, though the exact position of the IRES within the RhPV 5Ј-UTR has not been determined (47). These intriguing features of the RhPV 5Ј-UTR prompted us to undertake studies on the mode of function of its IRES.…”

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

“…IRES elements have also been found in the RNA genomes of other viruses including hepatitis C virus (HCV) and the pestiviruses (which are all members of the flaviviridae) plus the dicistroviridae, for example, cricket paralysis virus (Wilson et al 2000) and Rhopalosiphum padi virus (Woolaway et al 2001;Terenin et al 2005). Different types of viral IRES element have been shown to have distinct requirements for cellular proteins to allow translation initiation to occur efficiently (Doudna and Sarnow 2007).…”