To investigate the binding specificity of turnip mosaic virus (TuMV) viral protein-genome linked (VPg) with translation initiation factor 4E, we evaluated here the kinetic parameters for the interactions of human eIF4E, Caenorhabditis elegans IFE-3 and IFE-5 and Arabidopsis eIFiso4E, by surface plasmon resonance (SPR). The results indicated that TuMV VPg does not show a binding preference for Arabidopsis eIFiso4E, even though it is from a host species whereas the other eIF4E orthologues are not. Surprisingly, the effect of m(7)GTP on both the rate constants and equilibrium binding constants for the interactions of VPg differed for the four eIF4E orthologues. In the case of eIFiso4E and IFE-3, m(7)GTP increased k(on), but for eIF4E and IFE-5, it decreased k(on). To provide insight into the structural basis for these differences in VPg binding, tertiary structures of the eIF4E orthologues were predicted on the basis of the previously determined crystal structure of m(7)GpppA-bound human eIF4E. The results suggested that in cap-bound eIF4E orthologues, the VPg binds to the C-terminal region, which constitutes one side of the entrance to the cap-binding pocket, whereas in the cap-free state, VPg binds to the widely opened cap-binding pocket and its surrounding region. The binding of VPg to the C-terminal region was confirmed by the SPR analyses of N- or C-terminal residues-deleted eIF4E orthologues.