Abstract
The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (Ka) and average binding sites (n) for VPg were (8.51 ± 0.21) × 106 M–1 and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative ΔH°(–29.32 ± 0.13 kJmol–1) and positive ΔS° (36.88 ± 0.25 Jmol–1K–1). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m7G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 ± 1.27 and 75.37 ± 2.95 kJmol–1 respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.