The Triticum Mosaic Virus 5' Leader Binds to Both eIF4G and eIFiso4G for Translation.

Robyn Roberts,Aurélie M Rakotondrafara,Laura K Mayberry,Karen S Browning,Thomas Preiss

doi:10.1371/journal.pone.0169602

Robyn Roberts, Aurélie M Rakotondrafara + Show 3 more

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https://doi.org/10.1371/journal.pone.0169602

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Abstract

We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5’ untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5’ UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5’ UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

Highlights

For the majority of eukaryotic cellular mRNAs, translation initiation relies on the 5’ m7GpppG cap to recruit the necessary translation machinery
The main translation factor required for cap-dependent translation is the cap-binding complex, which is comprised of the smaller cap-binding protein subunit eIF4E that directly binds to the 5’ m7GpppG cap, the larger scaffold protein eIF4G which binds to eIF4E, and the helicase eIF4A [1]. eIF4E coordinates the attachment of the eIF4F complex to the RNA, and is recruited to the 5’ end of the mRNA via the 5’ cap [1]
We recently demonstrated that the TriMV bears a powerful translation element within its 5’ untranslated region (UTR) that has distinct features from other described plant viruses [18]

Summary

Introduction

For the majority of eukaryotic cellular mRNAs, translation initiation relies on the 5’ m7GpppG cap to recruit the necessary translation machinery. The main translation factor required for cap-dependent translation is the cap-binding complex (eIF4F), which is comprised of the smaller cap-binding protein subunit eIF4E that directly binds to the 5’ m7GpppG cap, the larger scaffold protein eIF4G which binds to eIF4E, and the helicase eIF4A [1]. EIF4E coordinates the attachment of the eIF4F complex to the RNA, and is recruited to the 5’ end of the mRNA via the 5’ cap [1]. The eIF4G/eIFiso4G isoforms share common binding motifs for the cap binding protein, eIF4A, and eIF3 in their C-terminal domains, but differ in PLOS ONE | DOI:10.1371/journal.pone.0169602. The cap-binding complex recruits the 43S ribosomal pre-initiation complex to the 5’ end of the mRNA via the eIF3-eIF4G interaction. Once the 43S subunit reaches the 5’ proximal AUG start codon, the 60S ribosomal subunit is recruited to the complex and translation initiates [1]

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