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Short Communication |
IPV, UMR GDPP INRA-Bordeaux 2, IBVM, BP 81, F-33883 Villenave d’Ornon Cedex, France
Correspondence
Olivier Le Gall
olegall{at}bordeaux.inra.fr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.
Present address: BIOGEMMA, 24 avenue des Landais, F-63170 Aubière, France. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), has received more attention since eIF4E defects were associated with an inability of several hosts to support potyvirus infection (Gao et al., 2004; Lellis et al., 2002; Nicaise et al., 2003; Ruffel et al., 2002). This suggested a biological significance for the eIF4E–VPg interaction, which was confirmed recently (Kang et al., 2005). In lettuce, eIF4E is encoded by mo1, a recessive resistance gene against Lettuce mosaic potyvirus (LMV) (Nicaise et al., 2003). VPg also interacts with other proteins encoded by the genome of the host (Dunoyer et al., 2004; Schaad et al., 1997; Yambao et al., 2003) or of the virus (Guo et al., 2001; Hong et al., 1995), including the multifunctional protein HcPro (Yambao et al., 2003). In several potyviruses, variations in the VPg central domain are associated with resistance-breaking (Borgstrøm & Johansen, 2001; Keller et al., 1998; Masuta et al., 1999; Moury et al., 2004; Nicolas et al., 1997; Rajamäki & Valkonen, 1999; Schaad et al., 1997). In this work, we investigated the interactions of LMV VPg, in particular its central domain, with lettuce eIF4E and LMV HcPro.
Recombinant lettuce (Lactuca sativa) eIF4E was expressed in bacteria from pDest17 (Invitrogen). Recombinant LMV VPg and HcPro were expressed in bacteria from pTrcHis (Invitrogen). These three proteins were purified from bacterial extracts as poly(His) fusions. In some experiments, His-tagged LMV HcPro purified from infected plants (Plisson et al., 2003) was used, yielding essentially similar results to those shown below (data not shown). Protein purity was controlled by SDS-PAGE (not shown). Rabbit polyclonal antibodies were obtained against VPg and eIF4E. Mouse monoclonal antibodies 1H5 and 21M were raised against VPg and VPg-CD (a synthetic peptide corresponding to the VPg central domain with a non-viral C-terminal extension, VFSDIGLVQDAFGKERLHAAAHAY), respectively. These mAbs were assessed by Western blotting and ELISA using infected lettuce or recombinant VPg (not shown); in addition, the reactivity of 21M for the VPg central domain was confirmed, as increasing concentrations of VPg-CD resulted in loss of ELISA signal (not shown). A monoclonal antibody against HcPro, 3C5, has been described previously (Roudet-Tavert et al., 2002). An ELISA-derived (Clark & Adams, 1977; Léonard et al., 2000) interaction assay was used. The surface was coated with eIF4E or HcPro [4 µg (ml carbonate buffer)–1 overnight at 4 °C] and saturated with fetal bovine serum (FBS; 30 min at room temperature) before incubation with VPg [8 µg (ml PBS/0.2 % Tween)–1, 0.2 % FBS for 1 h at 4 °C]. Interactions were revealed with 1H5 [5 µg (ml PBS/0.2 % Tween)–1 for 2 h at 37 °C], followed by antibodies conjugated to alkaline phosphatase. In reverse experiments, the wells were coated with VPg (4 µg ml–1) and the interaction with eIF4E or HcPro (8 µg ml–1) was revealed similarly using anti-eIF4E or 3C5.
A specific interaction was observed between VPg and eIF4E, as well as between VPg and HcPro (Fig. 1a). An N-terminally truncated version of HcPro (Plisson et al., 2003) also interacted with VPg (not shown), indicating that the first 99 N-terminal residues of HcPro are dispensable for the interaction with VPg. The interaction between eIF4E and VPg was confirmed by a spectrofluorometric approach, whereby binding of a ligand results in quenching of the fluorescence of eIF4E tryptophans. A decrease in eIF4E tryptophan fluorescence was observed in the presence of increasing concentrations of VPg (Fig. 2) but not of BSA used as a control (not shown). The data suggested a stoichiometry of one molecule bound per eIF4E and a dissociation constant in the same range as that of the cap–eIF4E interaction (Khan et al., 2006; Michon et al., 2006).
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), although not in Tobacco etch virus (TEV) (Schaad et al., 2000), amino acid changes in the VPg central region that impair eIF4E interaction are lethal, suggesting a direct interaction with eIF4E. The VPg central domain is also involved in HcPro binding (Yambao et al., 2003). Therefore, the involvement of the LMV VPg central domain in both HC-Pro and eIF4E interactions was assayed in vitro as described above. The anti-VPg-CD mAb 21M did not reveal an interaction between VPg and eIF4E (not shown), suggesting that the VPg central domain was not accessible to 21M once the VPg–eIF4E interaction had taken place and hence that this domain of VPg was involved directly in eIF4E interaction.
To test the hypothesis that eIF4E, HcPro and 21M bound in the same area on VPg, VPg was pre-incubated with 21M (1 : 3, w/w, for 2 h at 37 °C) and assayed for eIF4E binding. Such pre-treatment of VPg significantly diminished binding to eIF4E, as well as to HcPro (Fig. 1b
). When an anti-VPg polyclonal antibody was used for immunocapture of VPg in a control experiment, no reduction in signal was observed after pre-treatment, indicating that 21M did not interfere with recognition by 1H5, the mAb used to reveal the interaction. Taken together, this confirmed that 21M, when bound to the VPg central domain, interferes with further interaction with eIF4E or HcPro, and thus demonstrated that the central domain must remain available for specific interactions with eIF4E and HcPro to occur.
In order to confirm the localization of the interaction domain on VPg, the wells were coated with the synthetic peptide VPg-CD (5 µg ml–1) and incubated with eIF4E. The results were revealed using a mAb (2B12) directed against the N-terminal tag of the recombinant eIF4E. In a typical experiment, A405 values averaged 0.264±0.040 for VPg-CD compared with 0.028±0.006 for BSA, confirming a specific interaction. The interaction between eIF4E and VPg-CD was confirmed by spectrofluorometry, as above (Fig. 2), with the same dissociation constant as for VPg. Similarly, in a binding assay revealed using the anti-HcPro mAb 3C5, VPg-CD interacted with HcPro (A405=0.420±0.190 vs 0.028±0.025).
VPg-CD overlaps the region involved in the interaction between eIF4E and TuMV VPg (Léonard et al., 2000) and between HcPro and VPg of Clover yellow vein virus (Yambao et al., 2003). The present work therefore provides the first evidence that, for a single given potyvirus, HcPro and eIF4E bind to the same central domain of VPg (residues 89–105). This clearly suggests the possibility that they could compete for binding to VPg. In order to assess this possibility, we conducted an ELISA-based competition assay in which the effect of lettuce eIF4E on LMV HcPro–VPg binding was studied. In such experiments, the wells were coated with HcPro (5 µg ml–1) followed by VPg (1 µg ml–1) pre-incubated with either eIF4E or HcPro (1 h at 4 °C). In both cases, inhibition of HcPro–VPg interaction could be observed (Fig. 1c), confirming that HcPro and eIF4E compete for VPg binding.
A three-dimensional model of VPg from Potato virus Y has been predicted based on that of an unrelated protein showing a similar distribution of hydrophobic/hydrophilic residues (P
ochocka et al., 1996). VPg appeared as an egg-shaped protein, with the domain involved in the interaction exposed at one pole. Although this model was obtained through an unusual approach, it is consistent with an involvement in protein interactions.
To understand better the possible relationships between VPg function and structure, attempts were made to predict the folding of LMV VPg. Homology-based modelling was unsuccessful due to the lack of homologous proteins of known structure in databases (not shown). To predict the secondary structure of the interaction domain (Fig. 3), ten VPg sequences were aligned and the secondary structure was predicted for each of them using GOR-IV (Garnier et al., 1996). Other algorithms operated online (http://www.expasy.org and http://npsa-pbil.ibcp.fr) provided essentially similar results (not shown). A hydrophobic (Kyte & Doolitle, 1982) 
-sheet was returned for all sequences except those of bymoviruses, between the RNA-bound Tyr-64 (Murphy et al., 1991) and Asp-78, a residue associated with eIF4E-binding in TuMV (Léonard et al., 2000). For all viruses analysed, the downstream portion (residues 90–120) was predicted to be folded in a long amphiphilic -helix (Fig. 3). The amino acid variations associated with different biological properties (Borgstrøm & Johansen, 2001; Masuta et al., 1999; Moury et al., 2004; Nicolas et al., 1997; Rajamäki & Valkonen, 1999; Schaad et al., 1997), where identified unambiguously, mapped to the hydrophilic – presumably exposed – side of this helix, which is consistent with a role in protein interactions.
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ochocka et al., 1996), both are compatible with a surface exposition of the VPg central domain. Further structural and biochemical studies are needed to clarify the VPg modelling and the roles of the VPg central domain in the success of virus infection through interactions with eIF4E, HcPro and possibly other proteins. The biological implications of the duality of the interaction in which the VPg central domain is engaged with the host protein eIF4E and the virus protein HcPro will also require investigation. For instance, it has been established that changes in the VPg central domain that affect long-distance movement of TEV in tobacco are not necessarily associated with a loss of eIF4E interaction (Schaad et al., 2000). It is possible that this type of phenotype could be related to changes in affinity with HcPro, a viral protein involved in long-distance movement (Cronin et al., 1995).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Received 26 August 2006;
accepted 27 November 2006.
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) or VPg-CD (
). VPg or VPg-CD (0.05–5 µM) was added to a 2.5 µM eIF4E solution in 1 ml of 20 mM HEPES/KOH (pH 7.6), 25 mM KCl, 1 mM DTT, 10 % glycerol. The mixture was incubated (5 min at 25 °C) until a steady-state fluorescence at 342 nm was reached upon excitation at 280 nm. AU, arbitrary units. Inset: variation of eIF4E fluorescence as a function of increasing concentrations of VPg (