Báo cáo khoa học: 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 ppt

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:
Website: />(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
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 (K
d
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
m

7
GDP. 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 (K
c
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
m
7
GTP–Sepharose 4B is usually performed with free
m
7
GTP. We observed that, even after extensive dialysis,
a significant fraction of active eIF4E retains m
7
GTP
in its binding site (data not shown). In our study, all
eIF4E fractions were previously eluted from m
7
GTP–
Sepharose 4B with 1 m KCl instead of m
7
GTP (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-l

M VPg stock solution were added
to a 0.5-l
M 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 m
7
GDP 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
m
7
GDP 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 m
7
GDP. To discriminate between competitive and
noncompetitive interactions, we performed a m
7
GDP–
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 m
7
GDP binding to eIF4E at 25 °C. VPg
aliquots were mixed with 2 l

M eIF4E in buffer M Before titration
with m
7
GDP. Inset: isotherms of m
7
GDP 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)0.3l
M VPg; (n)1.2lM VPg; (h)2.4lM VPg.
Fig. 3. (A) Binding isotherms of m
7
GDP 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)30l
M VPg; (s)60lM 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 l
M.
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 K
app
c
can be derived

according to Scheme 1A as:
K
app
c
¼ K
c
1 þ
½V
free
K
v

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:
K
app
c
¼ K
c
K
v
þ½V
free
K

v
þ
½V
free
a
!
ð2Þ
where a and K
V
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–m
7
GDP, m
7
GDP–eIF4E–VPg,
and eIF4E–VPg whatever the concentration of
m
7
GDP, the plateau value is also affected and:
Y
app
sat
¼ Y
sat
K
v
aK

v
þ½V
free

ð3Þ
K
c
and Y
sat
were replaced in eqn (4) by the expressions
K
app
c
and Y
app
sat
. Assuming [V]
free
to be close to [V]
tot
,
data sets of eIF4E fluorescence as a function of
[m
7
GDP]
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 m

7
GDP
bind to distinct but interdependent sites (Fig. 3B).
Although the cap analogue and VPg displayed com-
parable affinity for eIF4E (K
c
¼ 0.31 ± 0.02 lm and
K
v
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 m
7
GDP 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
· K
a
(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

K
c
eIF4E
eIF4E-m
7
GDP
VPg
eIF4E-VPg
K
v
m
7
GDP
A
VPg-eIF4E-m
7
GDP
α
K
v
α
K
c
B
VPg
K
c
eIF4E
eIF4E-m
7

GDP
m
7
GDP
VPg
eIF4E-VPg
K
v
m
7
GDP
Scheme 1. Two plausible models for the interactions between
eIF4E, VPg and the cap analogue
7
mGDP. (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 GSTÆVPg
fusion (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 GSTÆ VPg 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
(K
d
¼ 0.083 ± 0.016 lm) was significantly higher than
that to the free eIF4E (K
d
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 m
7
GDP, 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 l
M, VPg final concentration 5 lM). Inset: (d) The apparent dis-
sociation constant K
d
app
of eIF4E–pep4G was determined in the
presence of increasing amounts of VPg. (s) The apparent dissoci-
ation constant K
d
app
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 m
7
GDP
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
C
18
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 QuickChange

TM
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 °Cinthe
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 A
600
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 Gateway
TM
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 A
280
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) ⁄ 1mm dithiothre-
itol was added to achieve a final concentration of 100 mm
KCl. This protein extract was incubated for 45 min at 4 ° C
with 2 mL m
7
GTP–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) ⁄ 1mm 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: e
280
¼
60 400 m
)1
Æcm
)1
and e

280
¼ 11 520 m
)1
Æcm
)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 l L 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 lg
GST 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 lL10mm 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 m
7
GDP 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,
1mm 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
F
cor
¼ F
obs
 10
ð0:5AexÞ
where F
cor
and F
obs
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 !
K
c
EC
According to this scheme, the saturation function of eIF4E
with its ligand follows an hyperbola:
½EC¼½E
tot
½C
K
c
þ½C
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 F

max
) 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 ¼
1 ) (F ⁄ F
max
). At ligand saturation, a maximum for Df
max
is
obtained, and we can define the plateau value as Y
sat
¼
1 ) (F
min
⁄ F
max
). Accordingly we can write:
½EC¼½E
tot
Y
Y
sat
ð5Þ
Eqn (1) becomes:
Y ¼ Y
sat
½C
K
c
þ½C

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 K
c
.
Eqn (7) relates the observed signal [Y ¼ 1 ) (F ⁄ F
max
)] to
[C]
tot
, the total ligand concentration [44]
Y ¼ K
c
þ½C
tot
þ Y
sat
ÀÁ
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
K
c
þ½C
tot
þ Y
sat

ÀÁ
2
À 4½C
tot
Y
sat
q
ð7Þ
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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