Mutational analysis of a plant cap-binding protein eIF4E reveals key amino-acids involved in

54 Pages·2008·1.56 MB·English

JVI Accepts, published online ahead of print on 14 May 2008
J. Virol. doi:10.1128/JVI.00209-08
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Mutational analysis of a plant cap-binding protein eIF4E reveals key
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2 amino-acids involved in biochemical functions and potyvirus infection.
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4 Running Title: Mutational analysis of eIF4E and potyvirus infection
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6 Sylvie German-Retana*, Jocelyne Walter, Bénédicte Doublet, Geneviève Roudet- w
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7 Tavert, Valérie Nicaise$, Cécile LecampiEon§, Marie-Christine Houvenaghel, Christophe ad
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8 Robaglia§, Thierry Michon and Olivier Le Gall fr
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10 Interactions Plante-Virus, UMR GDPP 1090, INRA Université de Bordeaux 2, BP 81, F- /jv
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11 A33883 Villenave d’Ornon Cedex, France m
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12 § UMR 6191 CEA-CNRS-Aix-Marseille Université, Laboratoire de Génétique et g/

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13 Biophysique des Plantes, Faculté des Sciences de Luminy, 13009, Marseille, France A
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14 $ Sainsbury laboratory, John Innes Centre, Norwich NR4 7UH UK il 5
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16 *Corresponding author: Sylvie German-Retana g
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17 UMR 1090 GDPP INRA Université Bordeaux 2 st
18 BP-81 F-33883 Villenave d’Ornon Cedex, France
19 Telephone +33.557.122.378
20 Fax +33.557.122.384, E-mail [email protected]
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22 Word count: abstract:215 ; text: 7334
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23 ABSTRACT
24 The translation initiation factor 4E (eIF4E, the cap-binding protein) is involved in
25 natural resistance against several potyviruses in plants. In lettuce, the recessive
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26 resistance genes mo1¹ and mo1² against Lettuce mosaic virus (LMV) are alleles coding
27 for forms of eIF4E unable or less effective to support virus accumulaEtion. A
28 recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome
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29 is able to produce symptoms in mo1¹ or mo1² varieties. In order to identify eIF4E
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30 amino-acid residues necessary for viral infection, we constructed recombinant LMV n
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31 expressing eIF4E with point mutations affecting various amino-acids and compared the d
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32 ability of these eIF4E mutaCnts to complement LMV infection in resistant plants. Three ro
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33 types of mutations were produced in order to affect different biochemical functions of t
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34 eIF4E: cap-binding, eIF4G binding, and putative interaction with other virus or host jv
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35 Aproteins. Several mutations reduced severely the ability for eIF4E to complement LMV m
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36 accumulation in a resistant host and impeded essential eIF4E functions in yeast. /
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37 However the ability for eIF4E to bind a cap analogue or to fully interact with eIF4G p
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38 appeared unlinked to LMV infection. In addition to providing a functional mutational ,

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39 map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be 9

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40 distinct from its physiological function in cellular mRNA translation. g
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41 INTRODUCTION
42 In the case of obligatory parasites such as viruses, the absence or inadequacy of a
43 single host factor may lead to the inability for the pathogen to multiply or to
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44 systemically invade its host (81, 84, 85). This implies that the dominant alleles of the
45 host genes involved would be associated with susceptibility and the recessivEe alleles
46 encoding non-functional versions of such host factors with resistance. In the case of
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47 potyviruses, it has been estimated that about 40% of the known resistance genes are
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48 recessive (58). n
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49 The genus Potyvirus is one of the largest and most diverse genera of plant viruses, and d
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50 causes severe losses to manCy crops, particularly vegetables (77). The flexuous potyvirus ro
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51 particles contain a single genomic RNA molecule of about 10000 nucleotides, encoding t
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52 a polyprotein which is matured by three virus-encoded proteinases (64). The genomic jv
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53 ARNA is polyadenylated at its 3’ end, and is not capped at its 5’ end but covalently m
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54 linked to a 25-kDa virus-encoded protein named VPg (50). Among host-encoded factors /
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55 required for potyvirus infection, the eukaryotic translation initiation factor eIF4E and/or p
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56 its isoform eIF(iso)4E have recently been demonstrated to play an important role. In ,

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57 Arabidopsis thaliana, disruption of the gene encoding eIF(iso)4E results in loss of 9

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58 susceptibility to at least four potyviruses, Tobacco etch virus (TEV), Turnip mosaic g
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59 virus (TuMV), Lettuce mosaic virus (LMV) (16, 35) and Plum pox virus (PPV) (14) and t
60 disruption of the gene encoding eIF4E compromises susceptibility to another potyvirus,
61 Clover yellow vein virus (73). In crop species, several natural recessive resistances
62 against potyviruses have been identified, and all were correlated to mutations in genes
63 encoding eIF4E (for review see 31, 66). The pvr2 locus in pepper, mo1 in lettuce, sbm1
64 in pea, confer resistance respectively against Potato virus Y (PVY) (69) and TEV (30),
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65 LMV (53), and Pea seed-borne mosaic virus (PSbMV) (18). The barley rym4/5/6
66 resistance genes to various strains of the bymoviruses (belonging to the family
67 Potyviridae) Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus
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68 (BaMMV), were also found to be mutations in the eIF4E gene (32, 78). Furthermore,
69 the nsv locus in melon that confers resistance to a non-potyviridae, uncapped aEnd non-
70 polyadenylated virus, Melon necrotic spot virus (MNSV) also encodes eIF4E (56).
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71 Simultaneous mutations in both eIF4E and eIF(iso)4E are required to prevent Pepper
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72 veinal mottle virus (PVMV) infection of pepper (70). Using the yeast two-hybrid system n
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73 and ELISA, it has been shown that the Viral Genome Linked protein (VPg) of TuMV d
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74 interacts with A. thaliana CeIF(iso)4E (80) and that a VPg mutation that abolishes this ro
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75 interaction is associated with a lack of infectivity of the derived TuMV in its natural t
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76 host Brassica (36). Yeam et al (82), recently showed that a critical substitution in the jv
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77 ACapsicum-eIF4E causing the loss of interaction with TEV-VPg is sufficient for the m
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78 resistance against TEV infection. Charron et al (11) recently provided evidence for co- /
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79 evolution between pepper eIF4E and potyviral VPg. The function of eIF4E and/or p
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80 eIF(iso)4E in the potyvirus cycle is still largely unknown, but the negative effect of ,

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81 natural mutations in these factors on the accumulation of various potyviruses in various 9

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82 host plants suggests that it is probably a conserved one (66). g
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83 In lettuce (Lactuca sativa), the only resistance genes currently used to protect lettuce
84 crops worldwide, are the recessive allelic genes mo11 and mo1². The mo11 gene,
85 formerly named g, was first identified in Argentina, in a Latin-type cultivar named
86 “Gallega de Invierno” (5) while the mo1² gene, identified in Egyptian wild L. sativa
87 lines was named mo (71). Initially considered identical, these genes were later shown to
88 have different specificities and to be either allelic or closely linked and therefore, were
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89 renamed mo11 and mo1² (15). They have been recently cloned and sequenced in our
90 laboratory (52). The resistance alleles mo11 and mo1², as well as the susceptibility allele
91 mo1°, were found to code for forms of the eukaryotic translation initiation factor Ls-
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92 eIF4E in lettuce. Types 0, 1 and 2 of lettuce eIF4E were named Ls-eIF4Eº, Ls-eIF4E¹
93 and Ls-eIF4E² respectively. Type 0 sequence corresponds to that found in suEsceptible
94 lettuce (Trocadéro, Salinas). Type 1 amino acid sequence (Mantilia and Floribibb) is
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95 characterized by a deletion of the triplet QGA at positions 108-110, replaced by an H
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96 residue. Type 2 sequence (Salinas 88 and Vanguard 75) has an A to P substitution at n
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97 position 70 (52). d
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98 The recessive alleles mo1¹ Cand mo1² at the mo1 locus are associated with reduced and ro
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99 symptom-less accumulation (tolerance) or absence of accumulation (resistance) of t
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100 common isolates of LMV (15, 19, 71). The issue of the interaction, resistance or jv
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101 Atolerance, depends on the virus isolate and genetic background (57, 65), but mo1¹ is m
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102 generally associated with resistance and mo1² with tolerance (9, 21). /
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103 In order to identify which eIF4E amino acids are important for the virus cycle, we set up p
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104 an experimental system by which the role of eIF4E in the virus cycle can be dissociated ,

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105 from its physiological function in cellular mRNA translation. It was previously 9

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106 observed that eIF4E from susceptible lettuce varieties (eIF4Eº), but not defective eIF4E g
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107 variants (eIF4E¹ and eIF4E² isolated from mo11 and mo1² varieties respectively) is able t
108 to restore LMV susceptibility when expressed ectopically in lettuce plants carrying
109 mo1¹ and mo1² (53).
110 We used this property to assay the effect of various amino-acid mutations on the
111 function played by eIF4E in the virus cycle. The mutations were chosen based on the
112 predicted tri-dimensional (3D) structure of lettuce eIF4E, so that they would affect
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113 amino-acids involved in cap recognition, eIF4G binding, and surface residues close to
114 the natural mutations associated with potyvirus resistance in lettuce mo1, pepper pvr2
115 and pea sbm1. The ability to complement LMV infection in mo1 plants should be
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116 affected for eIF4E mutated at amino-acids required for the virus cycle. Therefore, this
117 assay should allow an evaluation of the role of various structural and biocEhemical
118 properties of eIF4E in the potyvirus cycle.
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120 MATERIALS AND METHODS n
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121 Plant material e
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122 All lettuce (LactucaC sativa) plants were grown under standard greenhouse om

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123 conditions (16-h day length, 18 to 25°C) with additional light from 400 W sodium vapor tp
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124 pressure lamps, and maintained in insect-proof cages after inoculation. Genotypes with i.
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125 no mo1 resistance allele (Salinas and Trocadéro), carrying mo1¹ (Floribibb and .o
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126 Mantilia) and carrying mo1² (Salinas 88), were used in this study. Trocadéro was o
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127 routinely used to propagate LMV. The pair of lettuce genotypes included in this p
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128 analysis, Salinas/Salinas 88, is nearly isogenic for mo1² (27, 72). , 2
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129 Site-directed mutagenesis and viral constructs y

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130 The eIF4E coding sequence isolated from susceptible lettuce (eIF4E°) was cloned s
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131 into the vector pENTR/D-TOPO® (Invitrogen). This recombinant plasmid was used as
132 a template for PCR amplification to generate point mutations in the eIF4E sequence,
133 using the QuickChange® site directed mutagenesis kit (Stratagene). The mutations
134 introduced were W64A, F65A, W77L, R82L, E91A, Y113A, W123A, G156A, E157A,
135 R173A, A174P, W182A and S223L, where the first letter indicates the residue in the
136 wild type sequence, the figure gives the position of this amino-acid in the full-length
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137 lettuce eIF4E sequence and the second letter indicates the residue present in the mutated
138 protein.
139 Each of the mutant eIF4E cDNA was then cloned independently into an LMV-
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140 derived vector (20) in such a way that the recombinant LMV carries an insertion of the
141 eIF4E coding region in frame between the P1 and HcPro domains of LMVE, with a
142 NIaPro cleavage site resulting in the addition of eight amino-acids (PGDEVYHQ) at the
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143 C-terminus of eIF4E and five amino-acids (SDVPG) at its N-terminus after in vivo
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144 processing of the eIF4E protein from HcPro (53). In the non-recombinant LMV vector, n
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145 the introduction of an artificial NIa cleavage site leads to a HcPro with a slightly d
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146 modified N-terminus, whicCh does not affect its biological properties (20). The LMV ro
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147 isolate used, LMV-0, is unable to accumulate and produce symptoms in lettuce varieties t
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148 with the mo1¹ or mo1² gene, but insertion of the eIF4E cDNA in its genome (to obtain jv
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149 ALMV-4E°) restores full infectivity in such varieties (53). The non-recombinant LMV m
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150 vector and LMV-4E° were used as references throughout this work. /
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151 Lettuce plants (Trocadéro) primarily inoculated by biolistics (Helios Gene-Gun, p
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152 Bio-Rad, Hercules, CA) were homogenized in 25 mM Na HPO containing 2% ,
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153 diethyldithiocarbamate and used to rub-inoculate assay plants as previously described 9

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154 (63). The symptoms were monitored daily and the leaves were harvested for virus g
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155 titration 15 days post-inoculation (dpi), which is the time point when symptoms and t
156 virus accumulation in susceptible plants reach their maximum. The infection parameters
157 were compared within rather than across experiments. RT-PCR detection of the viral
158 progeny was performed as described previously (34), and the identity of each progeny
159 to the mutant inoculated was assessed by restriction and in some instances sequence
160 analysis. Semi-quantitative double-antibody sandwich ELISA was performed as
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161 described (20) after ten-times dilution of the plant extracts, so that the relationship
162 between A and antigen concentration was linear.
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163 Expression of wild-type and mutant eIF4E proteins in Escherichia coli D
164 Using the Gateway® Technology (Invitrogen), each of the mutated eIF4Es, as
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165 well as the wild-type, was transferred from pENTR/D-TOPO® into the pDEST™17
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166 destination vector, to allow production of N-terminal fusions with a 6xHis tag. The
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167 constructs were introduced into Escherichia coli (straiPn BL21-AI). Cells were grown at
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168 37°C to an A of 0.4 in 100 mL of LB medium containing ampicillin. The eIF4E lo
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169 expression was induced by addition of 0.2% arabinose, and continued over 3 hours at d

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170 30°C. The bacterial pellet was frozen and suspended in 4 mL HEX buffer pH 7.8 (20 m

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171 mM Hepes/KCOH, 1 mM DTT, 0.2 mM EDTA, 0.2 % Tween 20, 15% glycerol, 0.4 M p:
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172 NaCl). Lysozyme (0.5 mg/mL) and Phenyl-Methyl-Sulfonyl Fluoride (PMSF, 1 mM) i.a
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173 were added and the cell suspension was maintained for 45 min at 4°C with gentle o
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174 shaking. The suspension was sonicated and centrifuged (100000 g, 1 hour, 4°C), and the n

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175 supernatant was incubated for 45 min with 100 µL of Ni-NTA Sepharose CL-6B ril
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176 (Qiagen) equilibrated in HEX buffer. The beads were washed extensively with HEX 20
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177 containing 10 mM imidazole, until A reached a constant value. Proteins were eluted b
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178 by twice 200 µL of HEX containing 250 mM imidazole. The His-tagged protein e
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179 fractions were pooled and diluted in 1.2 mL of buffer A (20 mM Hepes-KOH pH 7.5, 1
180 mM DTT).
181 Expression and purification of wheat eIF4G will be published elsewhere (K.
182 Browning, personal communication).
183 Cap binding, eIF4G and VPg binding assays
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184 To evaluate their cap-binding abilities, the Ni2+-purified eIF4Es were subjected to
185 m7GTP-Sepharose affinity chromatography. After calibration of the protein
186 concentration, the proteins were incubated with 100 µL of m7GTP Sepharose 4B
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187 (Amersham biotech) at 4°C for 45 min. The beads were extensively washed with buffer
188 A (see above) and the proteins retained were eluted with 100 µL of 100 µM Em7GDP.
189 The fractions obtained were analyzed by western blotting using a rabbit polyclonal
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190 antibody raised against lettuce eIF4E.
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191 The eIF4G-binding ability of purified eIF4E was evaluated by fluorescence n
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192 spectroscopy using a synthetic peptide, pep4G (KKYSRDFLLKF), derived from A. d
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193 thaliana eIF4G (At3g6024C0, Genbank accession NP_567095), and including the 4E- ro
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194 binding motif YxxxxLΦ (39), where x represents any amino acid and Φ is an t
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195 hydrophobic amino acid. The fluorescence of tryptophans is sensitive to changes in their jv
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196 Aenvironment upon ligand binding. This feature was also exploited to monitor m
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197 quantitatively the interactions between various forms of purified eIF4E and their ligands /
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198 m7GTP and VPg0. All spectra were acquired at 25°C on a SAFAS Xenius p
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199 spectrophotometer (Monaco). The excitation wavelength was set to 280 nm. ,

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200 Measurements were made in 1 mL buffer A (see above) containing 100 mM KCl and 9

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201 10% glycerol. The concentration of eIF4E was set to 0.5 µM. Tryptophan fluorescence g
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202 quenching at 337 nm was monitored as a function of increasing amounts (0.005 to 5 t
203 µM) of ligand (either m7GTP, pep4G or VPg) in the mixture. For each concentration of
204 ligand added the fluorescence value retained was the mean value from a 2-minute
205 collection. Dissociation constants were deduced from data sets including at least three
206 independent titration experiments. Bovine serum albumin (Sigma) was used to control
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207 for non-specific binding. The dissociation constants were deduced by fitting the raw
208 data to the simple interaction model A + B ↔ AB (43).
209 An ELISA-derived interaction assay was used (12) to evaluate the ability of Ls-
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210 eIF4E proteins to bind the whole wheat 4G protein. Plates were coated with eIF4E
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211 proteins (wild type, E91A, W94A, G156A, E157A) diluted (5µg/ml) in carbonate buffer
212 (Na2CO3 15 mM, NaHCO3 35 mM). After an overnight incubationT at 4°C, plates were
213 washed and saturated with 10% Foetal calf serum in PBS (1h, room temperature) before
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214 incubation with wheat eIF4G (10µg/ml in PBS Tween SCF 0, 2%, 1h, 4°C). n
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215 Interactions were revealed with polyclonal antibodies against eIF4G (1/1000, 2h, 37°C). de
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216 Titrations of the coated pCroteins were assessed using polyclonal antibodies against ro
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217 eIF4E (1/1000, 2h, 37°C). Both titration and interactions were followed by anti-rabbit tt
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218 antibodies conjugated to Alkaline Phosphatase. An ELISA-derived interaction assay jvi.
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219 Awas also used to demonstrate the interaction of LMV VPg with Ls-eIF4E (68). This m
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220 assay was used to monitor the interaction of LMV VPg with Ls-eIF4E° and the surface /
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221 mutants. p
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222 Yeast complementation 0
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223 Saccharomyces cerevisiae strain J055 (cdc33-∆: LEU2 Leu2 ura3 his3 trp1 ade2 by

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224 [YCp33supex-h4E URA3]) contains a deletion of the chromosomal gene coding for e
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225 eIF4E and therefore its survival depends on the presence of plasmid YCp33supex-h4E
226 URA3 containing a copy of the human eIF4E cDNA, under the control of the glucose
227 repressible, galactose-dependent GAL promoter (26).
228 The cDNAs encoding each of the Ls-eIF4E mutant forms and the natural allelic forms
229 (4E°, 4E1 and 4E2) were independently transferred into the yeast-E. coli shuttle Trp-
230 selectable vector p426GPD (49) for expression in yeast under the control of the
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