Structural studies of the eIF4E–VPg complex reveal a direct competition for capped RNA: Implications for translation

Abstract

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5′ end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5′ end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg–eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E–eIF4G, eIF4E bound VPg–luciferase RNA conjugates, and these VPg–RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg–RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

A Novel Interaction Network Used by Potyviruses in Virus–Host Interactions at the Protein Level

“…Both our data and those from previous studies indicate that the central domain of VPg is required for the 4E interaction [8,11,[83][84][85]. Furthermore, our study indicates that PVA VPg contains a functional 4E-binding motif in the central part of VPg (aa89-95).…”

“…Although the cap-binding domain of 4E overlaps with the site of the VPg interaction, disrupted cap binding does not correlate with potyvirus resistance [11,15,16,79]. Instead, recent data indicate that VPg can compete with 4E for capped RNAs to inhibit host translation [11]. Plant eIF4Gs bind to 4E via their canonical 4E-binding motif but also via a second non-canonical motif consisting of hydrophobic residues, which connect to a dorsal and a lateral hydrophobic surface of 4E, respectively [18].…”

“…Previous studies demonstrate that 4E is involved in the nuclear export of selected mRNAs [75,76]. In addition, very recent data indicate that VPg can impair the nuclear export of 4E-dependent RNAs via binding to the cap-binding site and preventing RNA association with 4E in human cells [11]. Thus, the VPg-4E interaction might be the means by which potyviruses interfere with the nucleocytoplasmic trafficking of these host mRNAs to disrupt expression of some antiviral proteins.…”

“…Natural recessive resistance genes against different potyviruses encode 4E variants with amino acid substitutions near the cap-binding domain of 4E [8,14,[79][80][81][82]. Although the cap-binding domain of 4E overlaps with the site of the VPg interaction, disrupted cap binding does not correlate with potyvirus resistance [11,15,16,79]. Instead, recent data indicate that VPg can compete with 4E for capped RNAs to inhibit host translation [11].…”

“…In the many virus-host combinations in which the potyvirus is able to overcome 4E-mediated resistance, this is caused by mutations in the central part of VPg. One hypothesis suggests that VPg imitates the cap to support viral translation, and/or it inhibits host translation by sequestering 4E [8][9][10][11], the limiting factor for translation [12]. Consequently, the absence of an interaction between VPg and 4E would render the virus unable to complete its infection cycle.…”