Turnip Mosaic Virus Genome-Linked Protein VPg Binds C-Terminal Region of Cap-Bound Initiation Factor 4E Orthologue Without Exhibiting Host Cellular Specificity

Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka, Japan.
Journal of Biochemistry (Impact Factor: 2.58). 02/2009; 145(3):299-307. DOI: 10.1093/jb/mvn180
Source: PubMed


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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Available from: Robert Rhoads, Jan 18, 2015
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    • "Furthermore, the analysis of several resistant alleles in crops has made it possible to delimit regions I and II in the eIF4E1 protein sequence where AA substitutions involved in resistance to potyviruses tend to cluster [3]. It is possible to delimit region I and II in eIFiso4E because three-dimensional models suggest that eIF4E1 and eIFiso4E adopt a similar structure [24,25]. A higher degree of similarity was expected in regions I and II between Ca.eIF4E1 and At.eIFiso4E, but regions I and II are in fact much more conserved between At.eIF4E1 and Ca.eIF4E1 and between At.eIFiso4e and Ca.eIFIso4E, respectively (Figure 1B). "
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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.
    Full-text · Article · Mar 2014 · BMC Plant Biology
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    • "The interaction between eIF(iso)4E and the viral VPg has been proposed by several authors to participate in RNA stability , translation, or movement (Lellis et al. 2002; Okade et al. 2009). Additionally, eIF(iso)4E sequestration by VPg might impair translation of host mRNAs and promote viral RNA translation (Eskelin et al. 2011; Khan et al. 2008). "
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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.
    Full-text · Article · Dec 2012 · Molecular Plant-Microbe Interactions
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    • "Recently, eIF4E has been shown to interact physically with the potyviral VPg, as demonstrated using various protein– protein interaction assays, such as ELISA, yeast two-hybrid assays, bimolecular fluorescence complementation and surface plasmon resonance (Beauchemin et al., 2007; Charron et al., 2008; Léonard et al., 2000; Okade et al., 2009). Furthermore, this physical interaction was shown to correlate with successful viral infection of the plants (Beauchemin et al., 2007; Charron et al., 2008; Kang et al., 2005; Léonard et al., 2000; Yeam et al., 2007). "
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    ABSTRACT: Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1xAt-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.
    Full-text · Article · Oct 2009 · Journal of General Virology
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