ternary complex with the eukaryotic initiation factorseIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue Thierry Michon, Yannick Estevez, Jocelyne Walter, Sylvie German-R
Trang 1ternary 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 (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 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: michon@bordeaux.inra.fr
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 (Kd¼ 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.
Trang 2VPg 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 resistresist-ance 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 (Kd¼ 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 (Kc¼ 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-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.
Trang 3cap 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 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) 0.3 l MVPg; (n) 1.2 lM VPg; (h) 2.4 l M 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) 30 l M VPg; (s) 60 l M 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
Trang 4apparent dissociation constant Kapp
c can be derived according to Scheme 1A as:
Kcapp¼ Kc 1þ½Vfree
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:
Kcapp¼ Kc
Kvþ ½Vfree
Kvþ½Vfree
a
!
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:
Ysatapp¼ Ysat
Kv
aKvþ ½Vfree
ð3Þ
Kcand Ysatwere replaced in eqn (4) by the expressions
Kapp
c and Ysatapp 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 (Kc¼ 0.31 ± 0.02 lm and
Kv¼ 0.3 ± 0.03 lm), binding of the first molecule affected the binding of the second one by a factor 15 (a¼ 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 retainbind-ing 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.
Kc
VPg
eIF4E-VPg
Kv
m 7 GDP
A
VPg-eIF4E-m 7 GDP
αK v
αK c
B
VPg
Kc
m 7 GDP
VPg
eIF4E-VPg
Kv
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.
Trang 5contained 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.
Trang 6other 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 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 (Kd¼ 0.083 ± 0.016 lm) was significantly higher than that to the free eIF4E (Kd¼ 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.
Trang 7In 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 prelimintern-ary
titration of eIF4E with VPg up to saturation (eIF4E concentration
2 l M , VPg final concentration 5 l M ) 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 dappof 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.
Trang 8increase 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 Q
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
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
Mutagenesis Kit developed by Stratagene E coli (strain
BL21) was transformed with pTrcHisC-eIF4E or pTr-cHisC-VPg Overnight Luria–Bertani broth starters were
Luria–Bertani broth containing ampicillin were inoculated with 50 mL of the overnight culture starter and grown at
addition of 0.5 mm isopropyl b-d-thiogalactoside, and cells
DNA sequences encoding GST and GSTÆVPg fusion were
(Invitrogen) The expression vectors were introduced into
arabinose, the expression vector was run in the same condi-tions as above All proteins were submitted to the same
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
added, and the suspension was incubated for 45 min at
another 45 min The lysate was sonicated in ice for
3 min (1 s cycles) The crude extract was centrifuged at
buffer The beads were centrifuged at low speed and packed into a 2 mL column The beads were washed with HEX
con-stant value Proteins were eluted by a 10–250 mm imidazole
fractions containing proteins was pooled
Specific isolation procedures
After being desalted, VPg fractions were refined by
mono Q column (Amersham Biotech) Pure VPg was
recovered from ion metal affinity chromatography were
dithiothre-itol was added to achieve a final concentration of 100 mm
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
Trang 91 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,
Protein concentration was determined
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
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
super-natant was mixed with 100 lL glutathione–Sepharose 4B
beads, and 1 mL fractions were incubated with either 10 lg
recovered and washed extensively with ice-cold HEX buffer
The proteins were eluted with 100 lL 10 mm reduced
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
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
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
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
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
Fcor¼ Fobs 10ð0:5AexÞ
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:
Kc EC According to this scheme, the saturation function of eIF4E with its ligand follows an hyperbola:
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
Trang 10complex formation The association is followed by
For the sake of comparison between various sets of data,
Ysat
ð5Þ Eqn (1) becomes:
½C
An interesting feature of eqn (6) is that it expresses a
sat-uration function that does not require the true
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kcþ ½Ctotþ Ysat
4½CtotYsat
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
References
Description of Plant Viruses Association of Applied
Biologists, Wellesbourne
2 Riechmann JL, Lain S & Garcia JA (1992) Highlights
and prospects of potyvirus molecular biology J Gen
Virol 73, 1–16
3 Puustinen P, Rajamaki ML, Ivanov KI, Valkonen JP &
Makinen K (2002) Detection of the potyviral
genome-linked protein VPg in virions and its phosphorylation
by host kinases J Virol 76, 12703–12711
4 Murphy J, Rychlik W, Rhoads R, Hunt A & Shaw J (1991) A tyrosine residue in the small nuclear inclusion protein of Tobacco Vein Mottling Virus links the VPg
to the viral-Rna J Virol 65, 511–513
5 Murphy J, Klein P, Hunt A & Shaw J (1996) Replace-ment of the tyrosine residue that links a potyviral VPg
to the viral RNA Is lethal Virology 220, 535–538
6 Wen Y, Yue Z & Shatkin AJ (1998) Mammalian cap-ping enzyme binds RNA and uses protein tyrosine phosphatase mechanism Proc Natl Acad Sci USA 95, 12226–12231
7 Itoh N, Yamada H, Kaziro Y & Mizumoto K (1987) Messenger RNA Guanylyltransferase from Saccharo-myces cerevisiae Large scale purification, subunit func-tions, and subcellular localization J Biol Chem 262, 1989–1995
8 Pestova TV, Kolupaeva VG, Lomakin Ib, Pilipenko
EV, Shatsky IN, Agol VI & Hellen CU (2001) Mole-cular mechanisms of translation initiation in eukaryotes Proc Natl Acad Sci USA 98, 7029–7036
9 Schaad M, Anderberg R & Carrington J (2000) Strain-specific interaction of the tobacco etch virus Nla protein with the translation initiation factor elF4E in the yeast two-hybrid system Virology 273, 300–306
10 Wittmann S, Chatel H, Fortin M & Laliberte J (1997) Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4e of Arabidopsis thaliana using the yeast two-hybrid system Virology 234, 84–92
11 Leonard S, Plante D, Wittmann S, Daigneault N, Fortin
M & Laliberte J (2000) Complex formation between poty-virus VPg and translation eukaryotic initiation factor 4e correlates with virus infectivity J Virol 74, 7730–7737
12 Lellis AD, Kasschau KD, Whitham SA & Carrington
JC (2002) Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for Eif (iso) 4e during potyvirus infection Curr Biol 12, 1046–1051
13 Gao Z, Eyers S, Thomas C, Ellis N & Maule A (2004) Identification of markers tightly linked to sbm recessive genes for resistance to Pea seed-borne mosaic virus Theor Appl Genet 109, 488–494 Epub 6 April 2004
14 Nicaise V, German-Retana S, Sanjuan R, Dubrana MP, Mazier M, Maisonneuve B, Candresse T, Caranta C &
Le Gall O (2003) The eukaryotic translation initiation factor 4e controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus Plant Physiol 132, 1272–1282
15 Marcotrigiano J, Gingras AC, Sonenberg N & Burley
SK (1997) Cocrystal structure of the messenger RNA 5¢-cap-binding protein (eIF4E) bound to 7-methyl-Gdp Cell 89, 951–961
16 Carberry SE, Rhoads RE & Goss DJ (1989) A spectro-scopic study of the binding of m7GTP and m7GpppG
to human protein synthesis initiation factor 4e Bio-chemistry 28, 8078–8083