Turnip mosaic virus genome-linked protein VPg binds C-terminal region of cap-bound initiation factor 4E orthologue without exhibiting host cellular specificity.
ABSTRACT 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.
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ABSTRACT: The protein-protein interaction between VPg proteins of potyviruses and eIF4E or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected based on other previous results and comparison of protein models of cap-binding pockets and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine if these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of TuMV resistance of T1 and T2 transformants demonstrated that over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for engineering broad-spectrum resistance in Chinese cabbage.Molecular Plant Pathology 01/2014; · 3.88 Impact Factor
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ABSTRACT: Translation initiation factor eIF4E exerts an important role during infection of viral species in the family Potyviridae. Particularly, the eIF(iso)4E family member is required for Arabidopsis thaliana susceptibility to Turnip mosaic virus (TuMV), Lettuce mosaic virus (LMV) and Tobacco etch virus (TEV). In addition, a resistance mechanism named Restriction of TEV Movement (RTM) in A. thaliana controls the systemic spread of TEV in Col-0 ecotype. Here we describe that TEV-TAMPS, a Mexican isolate, overcomes the RTM resistance mechanism reported for TEV-7DA infection of the Col-0 ecotype, but depends on eIF(iso)4E for its systemic spread. To understand at which level eIF(iso)4E participates in A. thaliana TEV-TAMPS infection, the viral RNA replication and translation were measured. The absence or overexpression of eIF(iso)4E did not affect viral translation, and replication was still observed in the absence of eIF(iso)4E. However, the TEV-TAMPS systemic spread was completely abolished in the null mutant. The viral VPg precursor NIa was found in co-immunoprecipitated complexes with both, eIF(iso)4E and eIF4E. However, the viral coat protein CP was only present in the eIF(iso)4E complexes. Since both, the VPg and the CP proteins, are needed for systemic spread, we propose a role of A. thaliana eIF(iso)4E in the movement of TEV-TAMPS within this host.Molecular Plant-Microbe Interactions 12/2012; · 4.31 Impact Factor
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ABSTRACT: In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species. A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific. We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for plant breeding.BMC Plant Biology 03/2014; 14(1):67. · 4.35 Impact Factor