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

Figures

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  Fig 1. The TriMV 5’UTR RNA binds to eIF4G and eIFiso4G with greater affinity than the cap binding proteins. Bio-layer interferometry (BLI) was used to measure the binding kinetics between purified recombinant wheat eIF4G, eIFiso4G, eIF4E, or eIFiso4E protein and the viral 5’ UTR sequence (nt 1–739). Biotin-labeled RNA was bound to a streptavidin biosensor and applied to solutions containing different concentrations of proteins. A) Relative luciferase activity in wheat germ of a m7GpppG-capped and polyadenylated vector reporter in the presence of competing free RNAs in increasing molar excess A 0- to 20-fold molar excess of the competing free RNAs corresponding to the biotin-UTP labeled and unlabeled TriMV 5’UTR sequence (1–739) and the non-functional TriMV reverse sequence (739–1) were used. In B and C are shown the BLI sensograms revealing the binding curves for eIF4G/eIF4E (B) and eIFiso4G/eIFiso4E (C). A magnification of the binding curves for eIF4E and eIFiso4E is included. A table with their corresponding kinetics values is displayed below the appropriate graphs.

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  Fig 2. The TriMV 5’ UTR binds to eIF4G in the native protein translation complex. Biotin-UTP labeled RNAs were mixed with the ribosome salt wash (RSW) prior UV-crosslink and passed through a streptavidin column to capture the interacting factors. The TriMV 5’ UTR sequence (1–739), the TriMV reversed, non-functional sequence (739–1), a non-viral sequence control ((-) ctrl), the TriMV 5’ UTR sequence with a stem-loop structure disruption mutation at position 469–490 (ΔstemG>C), and the TriMV 5’ UTR with the double compensatory mutation (ΔstemC = G) were used as RNA baits. Western blot was used to detect each translation factor (eIF4E, eIF4G, eIFiso4E and eIFiso4G). A) Western blot against eIF4G, eIF4E, eIFiso4G and eIFiso4E of the ribosomal salt wash fractions and in wheat germ extract, relative to a fixed concentration (2 pmol) of each of the recombinant wheat factors. Using the Bio-Rad Image lab software, we approximated that eIFiso4G was present in 2.3x the concentration of eIF4G. B) Western blot against eIF4G, eIF4E, eIFiso4G and eIFiso4E proteins present in the ribosomal salt wash fraction and eluted from the biotinylated TriMV RNAs and controls. Unbound proteins obtained in the…

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  Fig 3. eIFiso4F can support TriMV 5’ UTR-mediated translation. A) Translation of ApppG-capped polyadenylated TriMV RNA in wheat germ extract depleted of cap-binding factors with increasing concentrations (0 to 10 pmol) of the wheat eIFiso4F complex or its individual subunits (eIFiso4G or eIFiso4E). The assay was performed with 14C-radiolabeled leucine, and the results display the incorporation of radiolabeled leucine (picomoles) in newly synthesized proteins following TCA protein precipitation. B) Western blot against the purified recombinant wheat eIF4G or eIFiso4G proteins individually eluated from the biotinylated TriMV RNAs and controls following UV-crosslink. Unbound proteins obtained in the flow-through and washes are shown. 0.25 pmol of each of the corresponding recombinant proteins was loaded in the first lane to serve as a ladder.

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  Fig 4. TriMV requires eIF4A helicase activity for its translation. A) Relative luciferase activity in wheat germ extract of the TriMV 5’ UTR and m7GpppG-capped control polyadenylated mRNAs with increasing concentrations of the eIF4A-inhibiting drug hippuristanol, suspended in 5% DMSO and added to the translation reaction. The luciferase activities were standardized to the measured luciferase activities of each mRNA with no hippuristanol added. At the 0 μM concentration of hippuristanol, equal volume of 5% DMSO alone also was added to the reaction. B) Translation inhibition by 10 μM hippuristanol can be reversed by adding 15 μM recombinant wheat eIF4A protein.