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Molecular and Cellular Biology, September 2005, p. 7879-7888, Vol. 25, No. 17
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.17.7879-7888.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia,¹ Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF, United Kingdom,² School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom³

Received 3 March 2005/ Returned for modification 5 April 2005/ Accepted 4 June 2005

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

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