The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation...

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The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue Thierry Michon, Yannick Estevez, Jocelyne Walter, Sylvie German-Retana and Olivier Le Gall Interactions Plante-Virus, UMR GDPP INRA-Bordeaux 2, Institut de Biologie Ve ´ge ´ tale Mole ´ culaire, Villenave d’Ornon, France Lettuce mosaic virus (LMV), is a member of the genus Potyvirus [1]. Potyviruses are plant viruses with flexible rod-shaped particles packing a single-stranded, poly- adenylated, positive-sense genomic RNA [2]. This RNA, of about 10 kb, is linked at its 5¢ end to a viral protein, VPg (virus protein linked to the genome), through a tyrosine phosphoester covalent bound [3–5]. The viral particle does not carry the molecular machin- ery required for its replication in the host cell, and the viral genome codes for a limited number of proteins. Thus the infectious cycle needs to recruit various host factors, including the host translation apparatus. Eukaryotic mRNAs are capped by the addition of a 7-methylated guanine (m 7 G). This post-transcriptional modification occurs in the nucleus [6,7]. The 5¢-cap acts as a flag on the mRNA for cytoplasmic export and for the ribosomal translation complex assembly. The recognition of this m 7 G functional group by a cap-binding protein (eIF4E, the eukaryotic translation initiation factor 4E, or its isoform eIFiso4E) is the first step of a complex cascade of molecular events leading to binding of the 40S ribosomal subunit to the mRNA [8]. The general structural similarity between the poty- virus RNA and eukaryotic mRNAs suggests that VPg may act functionally as a cap-like structure. This hypo- thesis gained strength when a specific interaction between eIF4E and VPg was identified in several patho- systems, such as tomato tobacco etch virus [9] and Arabidopsis thaliana turnip mosaic virus (TuMV) [10]. In the latter case, a single amino acid replacement in Keywords eIF4E; eIF4G; fluorescence; interaction; VPg Correspondence T. Michon, Virologie Ve ´ge ´ tale, GDPP, IBVM-INRA, BP 81, 33883 Villenave d’Ornon Cedex, France Fax: +33 5 57 12 23 84 Tel: +33 5 57 12 23 91 E-mail: [email protected] Website: http://www.bordeaux.inra.fr/ipv/ (Received 10 October 2005, revised 23 January 2006, accepted 26 January 2006) doi:10.1111/j.1742-4658.2006.05156.x The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5¢ end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap- binding protein of the eukaryotic translation initiation complex. We per- formed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m 7 GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K d ¼ 0.3 lm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruit- ment by VPg is indirectly mediated through VPg–eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions. Abbreviations BN, blue native; eIF4E, eukaryotic translation initiation factor 4E; GST, glutathione S-transferase; LMV, lettuce mosaic virus; PABP, poly(A)- binding protein; TuMV, turnip mosaic virus; VPg, virus protein linked to the genome. 1312 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

Transcript of The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation...

The potyviral virus genome-linked protein VPg forms aternary complex with the eukaryotic initiation factorseIF4E and eIF4G and reduces eIF4E affinity for a mRNAcap analogueThierry Michon, Yannick Estevez, Jocelyne Walter, Sylvie German-Retana and Olivier Le Gall

Interactions Plante-Virus, UMR GDPP INRA-Bordeaux 2, Institut de Biologie Vegetale Moleculaire, Villenave d’Ornon, France

Lettuce mosaic virus (LMV), is a member of the genus

Potyvirus [1]. Potyviruses are plant viruses with flexible

rod-shaped particles packing a single-stranded, poly-

adenylated, positive-sense genomic RNA [2]. This

RNA, of about 10 kb, is linked at its 5¢ end to a viral

protein, VPg (virus protein linked to the genome),

through a tyrosine phosphoester covalent bound [3–5].

The viral particle does not carry the molecular machin-

ery required for its replication in the host cell, and the

viral genome codes for a limited number of proteins.

Thus the infectious cycle needs to recruit various host

factors, including the host translation apparatus.

Eukaryotic mRNAs are capped by the addition of a

7-methylated guanine (m7G). This post-transcriptional

modification occurs in the nucleus [6,7]. The 5¢-cap

acts as a flag on the mRNA for cytoplasmic export

and for the ribosomal translation complex assembly.

The recognition of this m7G functional group by a

cap-binding protein (eIF4E, the eukaryotic translation

initiation factor 4E, or its isoform eIFiso4E) is the first

step of a complex cascade of molecular events leading

to binding of the 40S ribosomal subunit to the mRNA

[8]. The general structural similarity between the poty-

virus RNA and eukaryotic mRNAs suggests that VPg

may act functionally as a cap-like structure. This hypo-

thesis gained strength when a specific interaction

between eIF4E and VPg was identified in several patho-

systems, such as tomato ⁄ tobacco etch virus [9] and

Arabidopsis thaliana ⁄ turnip mosaic virus (TuMV) [10].

In the latter case, a single amino acid replacement in

Keywords

eIF4E; eIF4G; fluorescence; interaction; VPg

Correspondence

T. Michon, Virologie Vegetale, GDPP,

IBVM-INRA, BP 81, 33883 Villenave

d’Ornon Cedex, France

Fax: +33 5 57 12 23 84

Tel: +33 5 57 12 23 91

E-mail: [email protected]

Website: http://www.bordeaux.inra.fr/ipv/

(Received 10 October 2005, revised 23

January 2006, accepted 26 January 2006)

doi:10.1111/j.1742-4658.2006.05156.x

The virus protein linked to the genome (VPg) of plant potyviruses is a

25-kDa protein covalently attached to the genomic RNA 5¢ end. It was

previously reported that VPg binds specifically to eIF4E, the mRNAcap-

binding protein of the eukaryotic translation initiation complex. We per-

formed a spectroscopic study of the interactions between lettuce eIF4E and

VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg

bind to eIF4E at two distinct sites with similar affinity (Kd ¼ 0.3 lm). Adeeper examination of the interaction pathway showed that the binding of

one ligand induces a decrease in the affinity for the other by a factor of 15.

GST pull-down experiments from plant extracts revealed that VPg can

specifically trap eIF4G, the central component of the complex required for

the initiation of protein translation. Our data suggest that eIF4G recruit-

ment by VPg is indirectly mediated through VPg–eIF4E association. The

strength of interaction between eIF4E and pep4G, the eIF4E-binding

domain on eIF4G, was increased significantly by VPg. Taken together

these quantitative data show that VPg is an efficient modulator of eIF4E

biochemical functions.

Abbreviations

BN, blue native; eIF4E, eukaryotic translation initiation factor 4E; GST, glutathione S-transferase; LMV, lettuce mosaic virus; PABP, poly(A)-

binding protein; TuMV, turnip mosaic virus; VPg, virus protein linked to the genome.

1312 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

VPg abolished its interaction with eIF4E, and this

correlated with a defect in infectivity in Brassica

perviridis [11].

The simplest hypothesis is that VPg recruits eIF4E

to initiate the translation of the potyvirus polyprotein

[12]. The involvement of eIF4E in pea seed-borne

mosaic virus cell to cell movement has been suggested

[13]. Some lettuce cultivars display a recessive resist-

ance to LMV. As this resistance is opposed by the

eIF4E isoform, we focused this study on the VPg–

eIF4E interaction [14]. Whatever the biochemical pro-

cess involved, the VPg–eIF4E complex appears to play

a crucial role in the outcome of the plant–potyvirus

interaction. Therefore, we developed a quantitative test

to (a) measure precisely the binding strength between

VPg and eIF4E and (b) assess the pathway of the

interactions between VPg, eIF4E and the cap structure.

A glutathione S-transferase (GST) pull-down test from

plant extracts was used to demonstrate that VPg can

recruit the binary complex eIF4E–eIF4G, a component

of eIF4F, the complex of translation initiation. Finally,

we evaluated the possible effect of VPg on eIF4G

binding to eIF4E using a synthetic peptide that

mimicks the eIF4G-binding domain.

Results and discussion

Interaction between VPg and eIF4E

We initially evaluated the perturbation of the intrinsic

fluorescence of eIF4E upon its interaction with VPg.

As VPg contains no tryptophan, the contribution of its

intrinsic fluorescence was assumed to be negligible with

respect to the nine tryptophans present in eIF4E.

Upon the addition of VPg, a decrease in the overall

fluorescence of eIF4E was observed (Fig. 1). This

seemed to correlate with a specific interaction between

the two proteins, as the fluorescence decrease reached

a plateau at high VPg concentrations. The binding of

VPg only slightly affected eIF4E tryptophan fluores-

cence. The signal-to-noise ratio was poor, implying

low accuracy in the determination of the dissociation

constant (Kd ¼ 0.3 lm). The binding curve extrapola-

ted to a 1 : 1 stoichiometry for the two proteins

(Fig. 1 inset).

From these data it is likely that VPg binding to

eIF4E is not associated with much modification of the

environment of the eIF4E tryptophans. The two tryp-

tophan residues present in the cap-binding site are

accessible to the surface [15]. Direct access of VPg to

the pocket would probably be associated with greater

modification of the fluorescence of these two residues,

as happens when cap analogues penetrate into the

pocket [16]. The emission maximum of lettuce eIF4E

was found to be at 342 nm, which is rather high. It is

likely that the major contribution to the intrinsic fluor-

escence of the protein comes from these two trypto-

phans in a polar environment, the fluorescence of the

others being partially shielded. This was also found in

previous studies, although not to such an extent [16].

Binding-site characterization

To obtain a better fluorimetric response of eIF4E upon

its interaction with VPg, we attempted to follow indi-

rectly the complex formation using the cap analogue

m7GDP. There are considerable discrepancies between

the affinities of the cap analogue published so far. In the

absence of VPg, the value of the dissociation constant

obtained (Kc ¼ 0.31 ± 0.02 lm) was significantly lower

than in previous reports for this type of analogue

[17,18]. A possible explanation is that, during the proce-

dure used for eIF4E isolation, the elution step from

m7GTP–Sepharose 4B is usually performed with free

m7GTP. We observed that, even after extensive dialysis,

a significant fraction of active eIF4E retains m7GTP

in its binding site (data not shown). In our study, all

eIF4E fractions were previously eluted from m7GTP–

Sepharose 4B with 1 m KCl instead of m7GTP (see

Experimental Procedures). However, it cannot be exclu-

ded that lettuce eIF4E displays a higher affinity for the

Fig. 1. Fluorescence emission spectra of eIF4E upon VPg addition.

Aliquots of 2.5 lL from a 60-lM VPg stock solution were added

to a 0.5-lM eIF4E solution in buffer M. After each addition, the

mixture was incubated for 5 min at 25 �C, and spectra were recor-

ded. (- - - -) No VPg added; (—) from top to bottom increasing

amounts of VPg. Inset: variation in eIF4E fluorescence as a function

of VPg concentration in the medium.

T. Michon et al. Modulation of plant eIF4E properties by a potyvirus VPg

FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1313

cap than its wheatgerm counterpart. It is worth men-

tioning that, in a recent study, values in the nanomolar

range were reported. The authors emphasize that this

could be because the recombinant eIF4E used was

obtained from carefully renatured batches from inclu-

sion bodies [19], thus avoiding affinity purification

involving exposure to cap analogues.

The apparent dissociation constant of m7GDP to

eIF4E was higher in the presence of increasing

amounts of VPg (Fig. 2). The plateau value (ligand

saturation) was also affected (Fig. 2, inset). It was sus-

pected that VPg could remain bound to eIF4E even at

high cap analogue concentrations. The fact that

m7GDP could not displace VPg argued in favour of

the presence of a VPg-binding site on eIF4E, which is

distinct but structurally related to the cap-binding site.

A possible mixed-type noncompetitive ligand binding

of eIF4E and the cap to the VPg was reported previ-

ously [11]. However, as the amount of free and bound

ligand was not determined, a nonlinear double-recipro-

cal plot was obtained, from which it was difficult to

establish an accurate pathway for the interactions [20].

Complexes between proteins often involve large sur-

face overlaps. It cannot be ruled out that VPg interacts

with eIF4E through several domains, one of them

being structurally linked to the cap site. In such a case,

the presence of VPg might negatively affect the binding

of m7GDP. To discriminate between competitive and

noncompetitive interactions, we performed a m7GDP–

eIF4E binding test in the presence of a large excess of

VPg (30–60 lm) with respect to eIF4E (2 lm). In such

conditions, the concentration of free VPg ([V]free) is

assumed to be close to [V]total. The same saturation

behaviour was observed as for lower concentrations of

VPg (Fig. 3A). In the case of strict competition, the

Fig. 2. Effect of VPg on m7GDP binding to eIF4E at 25 �C. VPg

aliquots were mixed with 2 lM eIF4E in buffer M Before titration

with m7GDP. Inset: isotherms of m7GDP binding to eIF4E. Solid

lines represent theoretical data calculated using the best fit of the

experimental data to eqn (4) (see Experimental Procedures). (d) No

VPg; (s) 0.3 lM VPg; (n) 1.2 lM VPg; (h) 2.4 lM VPg.

Fig. 3. (A) Binding isotherms of m7GDP with eIF4E in the presence

of high VPg concentrations. Experimental conditions were as in

Fig. 2. Lines represent theoretical data calculated using the best fit

of the experimental data to eqn (4) using either two distinct but

dependent sites (solid line) or strict competition at the same site

(dashed line). All measurements were made at least in triplicate.

(d) 30 lM VPg; (s) 60 lM VPg. (B) Plots of residuals between the-

oretical and experimental data using either two distinct but depend-

ent sites (solid line) or strict competition at the same site (dashed

line). VPg concentration, 30 lM.

Modulation of plant eIF4E properties by a potyvirus VPg T. Michon et al.

1314 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

apparent dissociation constant Kappc can be derived

according to Scheme 1A as:

Kappc ¼ Kc 1þ ½V�free

Kv

� �ð1Þ

where [V]free is the concentration of free VPg in the

medium.

The apparent dissociation constant for binding at

two distinct but dependent sites defined in Scheme 1B

is:

Kappc ¼ Kc

Kv þ ½V�freeKv þ ½V�freea

!ð2Þ

where a and KV are the ratio of symmetrical dissoci-

ation constants (Scheme 1B) and the VPg–eIF4E disso-

ciation constant, respectively.

In this case, as eIF4E remains partitioned between

the three species eIF4E–m7GDP, m7GDP–eIF4E–VPg,

and eIF4E–VPg whatever the concentration of

m7GDP, the plateau value is also affected and:

Yappsat ¼ Ysat

Kv

aKv þ ½V�free

� �ð3Þ

Kc and Ysat were replaced in eqn (4) by the expressions

Kappc and Yapp

sat . Assuming [V]free to be close to [V]tot,

data sets of eIF4E fluorescence as a function of

[m7GDP]total were fitted to models mimicking either

strictly competitive binding or binding at two distinct

but dependent sites. Plots of residuals obtained from

both fits showed unambiguously that VPg and m7GDP

bind to distinct but interdependent sites (Fig. 3B).

Although the cap analogue and VPg displayed com-

parable affinity for eIF4E (Kc ¼ 0.31 ± 0.02 lm and

Kv ¼ 0.3 ± 0.03 lm), binding of the first molecule

affected the binding of the second one by a factor 15

(a ¼ 15 ± 3). We examined the effect of disruption of

the cap-binding capacity on eIF4E association with

VPg. To do so, we engineered W123A, an eIF4E

mutant in which W123, one of the two conserved tryp-

tophan residues involved in p-p stacking with the cap

aromatic moiety [15], was substituted with an alanine.

As expected, this substitution abolished m7GDP bind-

ing to W123A while retaining its capacity to associate

with VPg (Table 1). The VPg surface defining the zone

of interaction with eIF4E spans at least two distinct

domains, one of which can affect the topology of the

cap-binding pocket. The VPg–eIF4E interaction may

be a mechanism for recruiting the host translation

machinery cut off by several unrelated positive-stran-

ded RNA viruses. In a recent study, an interaction

between VPg and a human enteric calcivirus was dem-

onstrated [21].

GST pull-down assay of plant proteins forming

complexes with VPg

Several studies have reported specific interactions

between the VPg from potyvirus and eIF4E or one of

its isoforms [9]. A recombinant VPgÆGST fusion pro-

tein was used as a bait for trapping molecular species

susceptible to be recruited by VPg in planta. A soluble

protein fraction was recovered after mild detergent

treatment of the plant leaves. This extract was submit-

ted to a VPgÆGST pull-down procedure [22]. To deter-

mine the nature of the protein complexes involved in

specific interactions with VPg, the fraction recovered

was analysed by electrophoresis in native conditions. A

high-molecular-mass (above 200 kDa) species and two

minor species (below 100 kDa) were affinity-purified

from the protein extract (Fig. 4A, lane 2). A western

blot analysis with antibodies to VPg showed that all

Table 1. Equilibrium association constants for various forms of

eIF4E and its ligands. NB, no binding detected.

10)6 · Ka (M)1) VPg pep4G m7GDP

eIF4E 3.3 ± 0.3 4.1 ± 0.7 3.2 ± 0.3

eIF4E-VPg – 12.1 ± 1.5 0.2 ± 0.07

eIF4E-pep4G 2.9 ± 0.2 – 8.1 ± 0.9

W123A 1.5 ± 0.8 3.9 ± 0.4 NB

Kc

eIF4E eIF4E-m7GDP

VPg

eIF4E-VPg

Kv

m7GDPA

VPg-eIF4E-m7GDP

αKv

αKc

B

VPgKc

eIF4E eIF4E-m7GDP

m7GDP

VPg

eIF4E-VPg

Kv

m7GDP

Scheme 1. Two plausible models for the interactions between

eIF4E, VPg and the cap analogue 7mGDP. (A) Strictly competitive

model; (B) binding at two distinct but dependent sites.

T. Michon et al. Modulation of plant eIF4E properties by a potyvirus VPg

FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1315

contained this protein (Fig. 4A, lane 3). As the inter-

action of VPg with plant translation initiation factors

was suspected, a western blot was performed with spe-

cific antibodies to eIF4E (lane 4) and eIF4G (lane 5).

Two of the three species (the one >205 kDa and the

intermediate one) contained eIF4E (lane 4). The eIF4G

forms were restricted to the high-molecular-mass spe-

cies (lane 5). When pull-down assays were performed

on extracts from infected leaves, the highest-molecular-

mass species was not detected (lane 6).

The purified fraction was also analysed by

SDS ⁄PAGE to determine the composition of the com-

plexes. They mainly contained three groups of poly-

peptide chains according to their relative molecular

masses: a group with bands in the range of 180 kDa, a

54-kDa band, and a single chain of 26 kDa (Fig. 4B,

lane 4). The proteins were identified by western blot-

ting. Antibodies to eIF4G revealed many polypeptides

in the 70–200 kDa region (Fig. 4B, lane 5). It has pre-

viously been reported that, in several organisms,

eIF4G is highly susceptible to proteolysis [23]. It is

likely that, during the extraction step, cleavage

occurred along the polypeptide chain. Whereas blue

native (BN)-PAGE native conditions revealed a single

molecular species probably because of conformational

locking, SDS ⁄PAGE denaturing conditions revealed

the proteolysis.

The VPg antibodies reacted with a single 54-kDa

polypeptide corresponding to the GSTÆVPg fusion

(Fig. 4B, lane 6). The eIF4E antibodies revealed only a

26-kDa polypeptide in accordance with the expected

plant eIF4E (Fig. 4B, lane 7). Our GST pull-down

experiment showed that VPg can recruit eIF4E

(26 kDa [14]) and eIF4G (� 180 kDa according to

A. Thaliana eIF4G [24]). From the SDS ⁄PAGE

pattern we could determine precisely the nature of the

three complexes revealed by BN-PAGE analysis.

The highest-molecular-mass band (>205 kDa) corres-

ponds to the heterotrimer eIF4G–eIF4E–VPgÆGST

(� 260 kDa; Fig. 4A, compare lanes 3, 4 and 5).

The intermediate band (� 80 kDa) observed in native

conditions contained VPg and eIF4E but not eIF4G

(Fig. 4A, compare lanes 3 and 4 with 5). It was

attributed to the binary complex GSTÆVPg–eIF4E.The band of lowest molecular mass, � 54 kDa, on

BN-PAGE was unambiguously identified as the

GSTÆVPg fusion on SDS ⁄PAGE (Fig. 4B, lane 6; see

also lane 2 for comparison with pure GSTÆVPg).We demonstrate here that the VPg–eIF4E interac-

tion previously described in other pathosystems such

as TuMV ⁄B. perviridis is also found in the LMV ⁄ let-tuce system. However, this is the first report of a phys-

ical interaction between VPg and eIF4G. At this stage,

we found no evidence for a direct interaction between

VPg and eIF4G. The recruitment of eIF4G by VPg is

probably indirectly mediated by eIF4E as the central

element of the complex. This should display at least

two distinct binding sites, one for VPg and the other

for eIF4G. In control experiments, the protein extract

was incubated with unfused GST before the affinity

chromatography. GST alone failed to pull down any

A

B

Fig. 4. Analysis of the plant soluble proteins complexed with VPg.

(A) BN-PAGE of the protein fraction retained on glutathione–Seph-

arose 4b. Lane 2, the plant soluble extract was incubated with

recombinant VPgÆGst fusion and mixed with glutathione–Seph-

arose 4b beads. After extensive washing, the proteins specifically

retained on the resin were eluted with glutathione (see Experimen-

tal procedures for details). The fractions obtained were loaded on

to a 6–18% polyacrylamide slab gel. After migration, the complexes

were Coomassie stained. Proteins from lane 2 were transferred to

a nitrocellulose membrane. Complexes were revealed with poly-

clonal antibodies raised against VPg (lane 3), eIF4E (lane 4) and

eIF4G (lane 5). Lane 6, same as lane 2 except that protein extracts

were from LMV-Infected plants (40 lg VPgÆGst added). (B)

Sds ⁄ PAGE of the proteins retained on glutathione–Sepharose 4b.

Affinity chromatography was as in (A). Lane 4, electrophoretic pat-

tern of the protein fraction under denaturing conditions; Coomassie

blue staining. Lanes 5, 6 and 7: Western blot analysis of the pro-

teins retained using polyclonal antibodies raised against eIF4G, VPg

and eIF4E, respectively. Lane 1, molecular mass markers. Lane 2:

recombinant GSTÆVPg fusion extracted from E. coli and affinity-

purified on glutathione–Sepharose 4b. Lane 3, as a control, the plant

soluble extract was incubated with nonfused recombinant GST

and mixed with glutathione–Sepharose 4b. After being washed,

the fraction eluted with glutathione was analysed By SDS ⁄ PAGE.

Modulation of plant eIF4E properties by a potyvirus VPg T. Michon et al.

1316 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

other species from the plant extract, confirming that

the formation of specific complexes with eIF4E and

eIF4G only involved the VPg domain of the GSTÆVPgfusion (Fig. 4B, lane 3). In another set of experiments,

the GSTÆVPg pull-down was challenged by mixing

increasing amounts of pure recombinant VPg with the

plant extracts. The presence of VPg weakened the

interactions between the bait and eIF4E (Fig. 5A). To

study the interaction in the context of infection,

extracts from LMV-infected plants were incubated

with increasing amounts of purified GSTÆVPg before

affinity chromatography on glutathione–Sepharose 4B.

A 10-fold excess of GSTÆVPg was necessary to recover

an amount of eIF4E–GSTÆVPg complex comparable to

that observed from uninfected plants (Fig. 5B).

GSTÆVPg may be strongly challenged by the presence

of viral VPg forms involved in complexes with the ini-

tiation factors in planta. A very small amount of free

eIF4E may be accessible to GSTÆVPg, most of it asso-

ciated with the viral form. It was not possible to detect

the eIF4E–eIF4G complex from infected plant extracts

whatever the amount of GSTÆVPg added (Fig. 4A,

lane 6). If VPg binds strongly to the binary complex

eIF4E–eIF4G, it may hardly be displaced by

GSTÆVPg. Moreover, VPg can exist in several molecu-

lar forms in the infected cell. Sequential processing of

the polyprotein may be linked to a specific requirement

during each phase of the viral cycle. Of the molecular

forms of VPg, 6K2ÆVPgÆPro and VPgÆPro copurify with

eIF4E in complexes associated with membrane frac-

tions [25]. In infected cells, a substantial amount of

eIF4G may be involved in complexes with endogenous

forms of VPg. If these complexes are tightly bound to

membranes, they may be less efficiently extracted

under our conditions.

The strength of interaction between elF4E and

pep4G, the eIF4E-binding domain on eIF4G, is

enhanced by VPg

The eukaryotic eIF4F initiation complex is a hetero-

trimer consisting of eIF4E, eIF4A (an RNA helicase)

and eIF4G [26]. In mammals, the central part of

eIF4G contains three evolutionary conserved hydro-

phobic amino acids, a tyrosine and two consecutive

leucines separated by four less well conserved residues

(YxxxxLL). This motif is associated with eIF4E bind-

ing [27]. Small oligopeptides that mimick this eIF4E

recognition motif can bind to eIF4E. This recognition

motif is also highly conserved in the plant kingdom.

The lettuce eIF4G sequence is not available. Knowing

the high homology of this sequence between wheat

(Q03387) and A. thaliana (NP567095), the oligopeptide

KKYSRDFLLKF from A. thaliana (pep4G) was

synthesized and tested for its ability to bind to lettuce

eIF4E. In the mammalian 3D structure, Trp73 is in

close contact with the peptide [28]. Lettuce eIF4E was

modelled on the basis of its homology with the known

structure of its mammalian counterpart [14]. We hypo-

thesized that the fluorescence of Trp94 (Trp73 in

mouse) may be affected by pep4G binding. This fea-

ture has been used successfully to monitor pep4G

binding to murine eIF4E [19]. When eIF4E was incu-

bated either alone or after saturation with VPg, a

fluorescence decrease proportional to the amount of

complex formed was observed, leading to a saturation

plateau (Fig. 6). Interestingly the strength of binding

of pep4G to the preformed eIF4E–VPg complex

(Kd ¼ 0.083 ± 0.016 lm) was significantly higher than

that to the free eIF4E (Kd ¼ 0.24 ± 0.03 lm). The

reciprocal was not observed, as there was no effect of

increasing concentrations of pep4G on the binding

strength between VPg and eIF4E (Fig. 6 inset). As

found above for VPg and m7GDP, one could expect

the interdependence of VPg and pep4G with respect to

their binding to eIF4E. It cannot be ruled out that the

association of VPg with eIF4E induces a change in its

conformation, enhancing the fit of the eIF4E-binding

domain to pep4G. The interaction of VPg with eIF4E

affects the properties of both the cap-binding pocket

and the eIF4G-binding domain. Our observation is

consistent with VPg–eIF4E interactions mediated over

a large area. Long-range effects on the conformation

of eIF4E probably occur upon VPg binding. The data

should be interpreted with caution, as we cannot pre-

sume that the effect of VPg is the same for binding of

whole eIF4G. The association of pep4G with eIF4E

has been shown not to be accompanied by detectable

changes in the crystallographic structure of eIF4E [28].

Fig. 5. (A) Competition experiments with the recombinant VPg. The

plant soluble extract was incubated with recombinant VPgÆGst

fusion in the presence of 10 lg (lane 2) and 50 lg (lane 3) of pure

recombinant VPg; lane 1, no VPg added. After affinity chromatogra-

phy on glutathione–Sepharose 4b, the proteins were submitted To

BN-PAGE and analysed by western blotting with polyclonal anti-

eIF4E. (B) LMV-Infected plant extracts were incubated with increas-

ing amounts of recombinant GSTÆVPg fusion before affinity chroma-

tography. Proteins were analyzed by western blotting with

polyclonal antibodies against eIF4E. Lane 1, 10 lg VPgÆGst fusion;

lane 2, 30 lg VPgÆGst fusion; lane 3, 60 lg VPgÆGst fusion; lane 4,

100 lg VPgÆGst fusion; lane 5, 150 lg VPgÆGst fusion.

T. Michon et al. Modulation of plant eIF4E properties by a potyvirus VPg

FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1317

In fact, pep4G has a small effect on cap binding,

whereas whole eIF4G is a strong enhancer of eIF4E

binding to the mRNA cap structure [29]. A thermody-

namic analysis predicts that the cocrystal structure of

the pep4G–eIF4E complex encompasses most of the

energetically significant interactions between eIF4G

and eIF4E [28]. However, more recently, an NMR

structure of the binary complex between yeast eIF4E

and the eIF4G (393–490) domain was analyzed. It

shows unambiguously that the N-terminus of eIF4E,

which interacts with the eIF4G domain, promotes dis-

creet conformational changes in the cap-binding site,

allowing tighter binding of the cap [30]. The thermo-

dynamic parameters expressed as association constants

are tabulated for comparison (Table 1). VPg is an

efficient modulator of eIF4E properties. Scheme 2

summarizes a hypothetical distribution of the initiation

factors eIF4E and eIF4G according to the thermo-

dynamic binding strengths measured in this study.

In early stages of infection, the only form of VPg

present in the host cell is linked to the viral genome.

Its concentration is low compared with the concentra-

tion of host cap mRNAs. If VPg affinity in vivo is of

the same order of magnitude as in vitro, it is unlikely

that it will recruit eIF4E by displacing eIF4E–mRNA

complexes. Instead, the newly uncoated VPg–RNA

molecules will require free eIF4E to start translation

and ⁄or other steps of the virus cycle. It is likely that

the pull-down experiment on healthy plant extracts

reveals only eIF4E and eIF4G forms that are not

involved with cellular mRNA. As translation proceeds,

the amount of VPg increases in the infected cell. The

stoichiometry of the viral particle assembly is of the

order of 2000 capsid proteins for one VPg [31]. There-

fore, the synthesis and proteolytic maturation of the

polyprotein lead to a considerable excess of VPg over

that strictly required for virion assembly. According to

our data, the association of VPg with free eIF4E

should enhance its affinity for eIF4G. This would

account for the inability of the VPgÆGST fusion to dis-

place viral VPg. Most of the newly synthesized VPg

may recruit free eIF4E and possibly eIF4G, thereby

exerting a negative co-operative effect on the binding

of cellular mRNAs [32]. As eIF4E is the limiting factor

for translation efficiency [33], such sequestration may

affect host cell metabolism and perhaps contribute to

disease symptoms. A large amount of the VPg–Pro

form accumulates in the nucleus as nuclear inclusions,

in relation to a functional nuclear localization signal

present in the Pro domain [34]. In animal cells, a signi-

ficant proportion of eIF4E itself is located in the nuc-

leus [35]. If this is also true in plants, retention of

initiation factors in the nucleus through interaction

with nuclear VPg may contribute to depletion of the

cytoplasmic pool of free eIF4E, and disrupt mRNA

translation. In turn, the host cell may respond to this

disruption by increasing the expression of another

eIF4E isoform, as shown in B. perviridis after infection

with TuMV [25].

In eukaryotes, translation initiation of cellular

mRNAs is mediated through the closed loop model

connecting the capped 5¢ end of the messenger to its

polyadenylated 3¢ end. Circularization is thought to

Fig. 6. Effect of VPg on the association between eIF4E and pep4G.

(s) Titration of free eIF4E by pep4G. (d) The formation of the tern-

ary complex VPg–eIF4E–pep4G was monitored after a preliminary

titration of eIF4E with VPg up to saturation (eIF4E concentration

2 lM, VPg final concentration 5 lM). Inset: (d) The apparent dis-

sociation constant K dapp of eIF4E–pep4G was determined in the

presence of increasing amounts of VPg. (s) The apparent dissoci-

ation constant K dapp of eIF4E–VPg was determined in the presence

of increasing amounts of pep4G.

⇔VPg

eIF4E ⇔ eIF4E-cap

eIF4E-VPg cap-eIF4E-VPg

VPg

⇔eIF4G-eIF4E-cap

Scheme 2. Hypothetical pathways of eIF4E and eIF4G recruitment.

Large arrows highlight the connections that, according to the

experimental data, would be thermodynamically favoured.

Modulation of plant eIF4E properties by a potyvirus VPg T. Michon et al.

1318 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

increase translation efficiency by facilitating de novo

initiation and recycling of terminating ribosomes on

the same mRNA [36]. The 3¢ polyadenylated end of

mRNAs is bound to the poly(A)-binding protein

(PABP). Circularization of the potyvirus RNA could

be achieved through the 5¢ VPg instead of the cap

structure. The VPg–eIF4E–eIF4G–PABP complex

would bring together the 3¢ poly(A) and the 5¢ VPg

ends of the viral RNA. As it has been reported that

VPgÆPro from TuMV can bind to PABP [25], another

possibility is that circularization is achieved through

VPgÆPro–PABP, this more direct binding skipping the

eIF4G–PABP interaction [37]. Both mechanisms may

be present at different stages of the virus cycle. It is

likely that viral recruitment of the 40S ribosome sub-

unit is mediated by the eIF3–eIF4G–eIF4E interaction

as for cellular mRNAs (see [38] for a review of trans-

lation initiation factors). Although there is no evident

internal ribosome entry site in the 5¢ part of the LMV

genome, the possibility of internal positioning of the

ribosome was demonstrated using an uncapped

tobacco etch virus 143 nucleotide leader. It has been

shown that, in such conditions, translation still

requires eIF4G [39]. Taken together, the data presen-

ted here strengthen the hypothesis of a physical

involvement of eIF4G in the translation initiation

complex of LMV.

Experimental Procedures

Materials

Desalted water was further purified using a Milli-Q Milli-

pore system. Buffers, salts (reagent grade), and m7GDP

were from Sigma Aldrich (Lyon, France). All solutions

were filtered through a 0.22-lm membrane before use. The

peptide pep4G was synthesized by a solid-phase method

using F-moc chemistry [40] and purified by reversed-phase

C18 HPLC. Polyclonal antibodies were raised in rabbits

after injection with purified recombinant proteins.

Protein expression and isolation

The gene coding for the eIF4E was cloned from the lettuce

(Lactuca sativa) cultivar Salinas AAP86602, which is sus-

ceptible to LMV [14]. The VPg coding sequence was PCR-

amplified from a partial LMV cDNA (isolate LMV-0,

P31999) cloned in Escherichia coli. Both cDNAs were

inserted into the pTrcHis-C expression vector (Invitrogen)

in-frame with an N-terminal hexahistidine tag, according to

the manufacturer’s instructions. The W123A mutant of

eIF4E was built using the QuickChangeTM Site-Directed

Mutagenesis Kit developed by Stratagene. E. coli (strain

BL21) was transformed with pTrcHisC-eIF4E or pTr-

cHisC-VPg. Overnight Luria–Bertani broth starters were

inoculated with single colonies and cultured at 37 �C in the

presence of 50 lgÆmL)1 ampicillin. Larger volumes (2 L) of

Luria–Bertani broth containing ampicillin were inoculated

with 50 mL of the overnight culture starter and grown at

37 �C to reach A600 of 0.5. Gene expression was induced by

addition of 0.5 mm isopropyl b-d-thiogalactoside, and cells

were incubated at 30 �C with shaking for another 3 h.

DNA sequences encoding GST and GSTÆVPg fusion were

cloned in pDEST 15 according to the GatewayTM strategy

(Invitrogen). The expression vectors were introduced into

E. coli (BL21AI strain). After induction with 2 lgÆmL)1

arabinose, the expression vector was run in the same condi-

tions as above. All proteins were submitted to the same

extraction procedure and kept at 4 �C. Phenylmethanesulfo-

nyl fluoride (100 lL of a 200-mm stock solution in meth-

anol) was added at each step of the extraction. Cells were

centrifuged and suspended in 30 mL HEX buffer (20 mm

Hepes ⁄KOH, pH 8, 1 mm dithiothreitol, 250 lm EDTA,

0.25% Tween 20, 0.4 m KCl). Lysozyme (0.5 mgÆmL)1) was

added, and the suspension was incubated for 45 min at

4 �C with gentle stirring. DNase and RNase (100 lg each)

were added and the suspension was incubated at 4 �C for

another 45 min. The lysate was sonicated in ice for

3 min (1 s cycles). The crude extract was centrifuged at

20 000 g, 4 �C for 30 min. The supernatant was recovered

and centrifuged at 100 000 g, 4 �C for 45 min. The clarified

supernatant was incubated for 45 min with 2 mL Ni ⁄nitrilo-triacetate ⁄ Sepharose CL-6B (Qiagen) equilibrated in HEX

buffer. The beads were centrifuged at low speed and packed

into a 2 mL column. The beads were washed with HEX

containing 10 mm imidazole until the A280 reached a con-

stant value. Proteins were eluted by a 10–250 mm imidazole

step gradient (1 mLÆmin)1 flow rate). The eluted fractions

were submitted to SDS ⁄PAGE, and the material from the

fractions containing proteins was pooled.

Specific isolation procedures

After being desalted, VPg fractions were refined by anion-

exchange chromatography in 20 mm Tris ⁄HCl, pH 8, on a

mono Q column (Amersham Biotech). Pure VPg was con-

centrated and stored in 50% glycerol at )20 �C until use

(yield � 10 mg per litre of culture). The eIF4E fractions

recovered from ion metal affinity chromatography were

pooled, and 20 mm Hepes ⁄KOH (pH 7.5) ⁄ 1 mm dithiothre-

itol was added to achieve a final concentration of 100 mm

KCl. This protein extract was incubated for 45 min at 4 �Cwith 2 mL m7GTP–Sepharose 4B (Amersham Biotech)

equilibrated in buffer A (20 mm Hepes ⁄KOH, pH 7.5,

100 mm KCl, 1 mm dithiothreitol). The beads were centri-

fuged at low speed, packed into a 2-mL column, and

washed with buffer A. Protein elution was performed with

T. Michon et al. Modulation of plant eIF4E properties by a potyvirus VPg

FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS 1319

1 m KCl in buffer A. After SDS ⁄PAGE analysis, the

fractions containing eIF4E were pooled and concentrated

under nitrogen on an Amicon ultrafiltration cell equipped

with a YM10 membrane (Millipore). After being desalted,

1-mL aliquots (0.5 mgÆmL)1) were recovered in 20 mm

Hepes ⁄KOH (pH 7.5) ⁄ 1 mm dithiothreitol ⁄ 20 mm KCl

and stored at )20 �C (yield 1–2 mg per litre of culture).

Protein concentration was determined spectrophotometri-

cally using the following absorption coefficients: e280 ¼60 400 m

)1Æcm)1 and e280 ¼ 11 520 m)1Æcm)1 for eIF4E and

VPg, respectively. GST and GSTÆVPg fusions were affinity-

purified on glutathione–Sepharose 4B beads (Amersham

Bioscience) as described in the following section.

Pull-down assays

Fresh leaves (1 g) were frozen in liquid nitrogen and

ground. The powder was suspended in 2 mL cold HEX

buffer. Then 10 lL 200 mm phenylmethanesulfonyl fluoride

stock solution in methanol and 25 lL Sigma P8340 prote-

ase cocktail inhibitor were added. The suspension was fil-

tered through glass wool. The filtrate was centrifuged at

10 000 g for 10 min at 4 �C. Phenylmethanesulfonyl fluor-

ide and protease cocktail inhibitor were added. Typically

2.5 mg soluble proteins were recovered by this procedure.

The extract was incubated with 100 lL glutathione–Seph-

arose 4B beads and 25 lg GST for 2 h at 4 �C. The super-

natant was mixed with 100 lL glutathione–Sepharose 4B

beads, and 1 mL fractions were incubated with either 10 lgGST or 20 lg GSTÆVPg fusion for 2 h at 4 �C. After cen-

trifugation at 10 000 g for 2 min, 4 �C, the beads were

recovered and washed extensively with ice-cold HEX buffer.

The proteins were eluted with 100 lL 10 mm reduced gluta-

thione in 20 mm Hepes ⁄NaOH, pH 8. Aliquots of volume

10 lL were used for PAGE analysis. BN-PAGE was car-

ried out using 6–18% polyacrylamide slab gels as described

previously [41]. Proteins were revealed by western immuno-

blotting with specific polyclonal antibodies.

Fluorescence measurements

All spectra were acquired at 25 �C on a SAFAS Xenius

spectrophotometer (Monaco). Excitation and emission slits

were set to a 10-nm path. The photomultiplier’s power was

set at 600–800 V (of a maximum possible value of 1200 V).

The photons emitted were collected at right angles from the

vertical excitation beam. The geometry of the device

allowed us to set the optical path length of the emitted light

1 mm above the excitation source. With this set-up, fluores-

cence emission was linear up to 0.7 absorbance units at the

wavelength of excitation (data not shown). The excitation

wavelength was set at 258 nm. Although there is a contri-

bution of m7GDP absorption at this wavelength, with our

optical system the inner filter effect was about 2% at the

highest concentration and no correction was made. By exci-

ting the sample at 258 nm, the Raman peak contribution to

the emission spectrum was prevented. In water, Raman

scattering occurs � 30 nm above the excitation wavelength.

In the buffer used, the excitation at 258 nm was accompan-

ied by a Raman peak at 282 nm, which was far from the

maximum of emission observed (343 nm). The nine trypto-

phan residues in eIF4E make the intrinsic fluorescence

important, allowing the recovery of a very good signal even

when exciting at 258 nm.

These conditions proved in preliminary experiments to

give the best ratio of signal to noise for the observation of

tryptophan fluorescence quenching at 342 nm. This set-up

was in accordance with the original studies of eIf4E titra-

tion with cap analogues [16,42]. Measurements were made

in 1 mL buffer M (20 mm Hepes ⁄KOH, pH 7.6, 25 mm Kcl,

1 mm dithiothreitol and 10% glycerol). The concentration

of eIF4E was between 0.5 and 2.5 lm. Ligand stock solu-

tions were adjusted in such a way that the volume added

upon titration never exceeded 5% of the total volume.

After addition of the ligand, emission at 342 nm was recor-

ded over a period suitable for reaching a constant fluores-

cence value (steady-state usually after 5 min). The decrease

in fluorescence with respect to the initial fluorescence was

recorded, and corrections were made for dilution. BSA was

used as a control under the same conditions to test for

nonspecific binding. The fluorescence signal did not show

significant changes upon BSA Addition (data not shown).

In some experiments, it was necessary to perform emission

measurements in the presence of high VPg concentrations.

Quantum yield loss was estimated according to the equation

Fcor ¼ Fobs � 10ð0:5AexÞ

where Fcor and Fobs are the intensity of fluorescence correc-

ted and observed, and Aex is the absorbance of the solution

at 258 nm [43]. As the quantum yield decrease was less than

3%, no inner filter correction was necessary. VPg emission

was checked at high VPg concentration. No significant light

scattering was observed. For each ligand concentration,

three data acquisitions were made.

Data analysis

Let us consider the simple bimolecular association between

eIF4E and one ligand:

E þ C !Kc

EC

According to this scheme, the saturation function of eIF4E

with its ligand follows an hyperbola:

½EC� ¼ ½E�tot½C�

Kc þ ½C�ð4Þ

The experimental procedures used in this work do not

allow direct determination of bound and free ligand

concentrations without access to [Ec] and [C]. eIF4E

fluorescence decreases together with the eIF4E–ligand

Modulation of plant eIF4E properties by a potyvirus VPg T. Michon et al.

1320 FEBS Journal 273 (2006) 1312–1322 ª 2006 The Authors Journal compilation ª 2006 FEBS

complex formation. The association is followed by monitor-

ing Fmax ) F as a function of the amount of ligand added.

For the sake of comparison between various sets of data,

the fluorescence decrease was normalized to Y ¼1 ) (F ⁄Fmax). At ligand saturation, a maximum for Dfmax is

obtained, and we can define the plateau value as Ysat ¼1 ) (Fmin ⁄Fmax). Accordingly we can write:

½EC� ¼ ½E�totY

Ysatð5Þ

Eqn (1) becomes:

Y ¼ Ysat½C�

Kc þ ½C�ð6Þ

An interesting feature of eqn (6) is that it expresses a sat-

uration function that does not require the true concentra-

tion of the active protein ([E]tot) in the sample to be known

to determine Kc.

Eqn (7) relates the observed signal [Y ¼ 1 ) (F ⁄Fmax)] to

[C]tot, the total ligand concentration [44]

Y ¼ Kc þ ½C�tot þ Ysat

� ��

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiKc þ ½C�tot þ Ysat

� �2 � 4½C�totYsat

qð7Þ

Hypothetical models of interaction were examined by fitting

the associated equations to experimental data using non-

linear regression. Affinity constants were determined from

the model giving the best fit to the data.

Acknowledgements

We would like to thank Genevieve Roudet for her skil-

ful assistance, and Dr T. Candresse and Dr T. Delau-

nay for stimulating discussions. Many thanks to

Professor K. S. Browning for providing us with eIF4G

polyclonal antibodies. We thank Dr J. F. Bussotti

from SAFAS S.A. for his skilful assistance in optical

device optimization. We are grateful to C. Manigand

(UMR 5471 CNRS-Bordeaux 1) for pep4G synthesis.

We are indebted to region Aquitaine for its financial

support for this work.

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