Abstract

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

Highlights

  • All plant and vertebrate viruses are parasites of the host’s translational machinery because they do not encode any of its components [1]

  • We found a wide range in efficiency of translation facilitated by different panicum mosaic virus-like CITE (PTE), ranging from background levels, to half that conferred by one of the most active mammalian virus IRESes known

  • Under non-saturating mRNA concentrations, at short time points at which the rate of translation is most rapid and sensitive to differences in efficiency, we observed wide variation in efficiencies of translation mediated by the different PTEs in monocot and dicot systems

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Summary

Introduction

All plant and vertebrate viruses are parasites of the host’s translational machinery because they do not encode any of its components [1]. Instead of an IRES, many plant viruses contain a sequence in their 30 UTR, which allows efficient cap-independent translation initiation via ribosome scanning from the 50 end of the genome [12,13,14] These 30 cap-independent translation elements (30 CITEs) bind a surface of translation initiation factor heterodimer eIF4F with high affinity and, in most cases, are thought to deliver this and possibly other factors and the 40S ribosomal subunit, to the 50 end via long-distance base pairing between the 30 and 50 UTR [15,16,17,18,19,20,21]. The data shown are averages of triplicates from two experiments and error bars represent standard error

Results
The TPAV PTE Binds Human eIF4E
G U UUGU A C
G Domain
G Domain C G G 4050
Interaction of Mammalian eIF4E with TPAV PTE
Materials and Methods
Plasmid Constructs and RNA Synthesis
Recombinant Protein Expression
Translation in Plant Systems
Translation in the Mammalian Systems
RNA Structure Probing and Footprinting

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