Here, we examined the binding of Lproto eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645–657 of human eIF4GI.. Comparison of these amino
Trang 1proteinase to eIF4GI involves conserved ionic interactions Nicole Foeger*, Elisabeth Kuehnel†, Regina Cencic and Tim Skern
Max F Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Austria
The eukaryotic translation initiation factor (eIF) 4F is
a protein complex that mediates recruitment of
ribo-somes to mRNA [1] This event is one of the
rate-lim-iting steps for translation and thus an important target
for translational control The eIF4F complex consists
of several components: eIF4E, a protein recognizing
the 5¢ cap structure of the mRNA; the RNA helicase
eIF4A; and the bridging protein eIF4G, that brings
together mRNA and ribosome via mRNA
circulariza-tion [2] eIF4G is a central part of this complex as it
provides binding sites not only for the already men-tioned translation factors eIF4E and eIF4A [3], but also for the ribosome-associated eIF3 [4], the poly(A) binding protein [5] and the eIF4E kinases Mnk 1 [6] and Mnk 2 [7]
Picornavirus infection leads to the so-called host cell shut-off Virally encoded picornaviral proteases cleave eIF4GI and eIF4GII, thereby leading to an inhibition
of cap-dependent cellular protein synthesis [8,9] Viral translation is unaffected as it initiates via an internal
Keywords
Foot-and-mouth disease virus; papain-like
proteinase; self-processing; exosite; protein
synthesis inhibition
Correspondence
T Skern, Max F Perutz Laboratories,
University Departments at the Vienna
Biocenter, Department of Medical
Biochemistry, Medical University of Vienna,
Dr Bohr-Gasse 9 ⁄ 3, A-1030 Vienna, Austria
Fax: +43 14277 9616
Tel: +43 14277 61620
E-mail: timothy.skern@meduniwien.ac.at
Website: http://www.meduniwien.ac.at/
medbch
Present addresses
*Division of Cell Biology and †Division of
Tumour Genetics, German Cancer Research
Center, Im Neuheimer Feld 280, D-69120
Heidelberg, Germany
(Received 22 February 2005, revised 20
March 2005, accepted 24 March 2005)
doi:10.1111/j.1742-4658.2005.04689.x
The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) ini-tially cleaves itself from the polyprotein Subsequently, Lpro cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still trans-lated, initiating from an internal ribosome entry site Lpro cleaves eIF4GI between residues G674 and R675 We showed previously, however, that
Lpro binds to residues 640–669 of eIF4GI Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay Lprointeracts with eIF4GI via resi-due C133 and resiresi-dues 183–195 of the C-terminal extension This binding domain lies about 25 A˚ from the active site Here, we examined the binding
of Lproto eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645–657 of human eIF4GI Comparison of these amino acids with those in human eIF4GII as well as with sequences
of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650) Mutation of these residues was severely detri-mental to Lprobinding Similarly, comparison of the sequence between resi-dues 183 and 195 of Lprowith those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved Substitution of these residues in Lpro significantly reduced eIF4GI binding and cleavage without affecting self-processing Thus, FMDV Lpro has evolved a domain that specifically recognizes a host cell protein
Abbreviations
2Apro, 2A proteinase; CTE, C-terminal extension; eIF, eukaryotic initiation factor; ERAV, equine rhinitis A virus; ERBV, equine rhinitis B virus; FMDV, foot-and-mouth disease virus; HRV, human rhinovirus; L pro , leader proteinase, containing amino acids 1–201; Lb pro , shorter form of
L pro containing amino acids 29–201; RRL, rabbit reticulocyte lysate.
Trang 2the remainder of the complex is sufficient for viral
translation
FMDV Lpro is the most N-terminal protein on the
viral polyprotein, the primary translation product
pro-duced from the viral RNA genome As a papain-like
cysteine proteinase, Lpro shows a typical two domain
a-helix⁄ b-sheet fold of a papain proteinase but is
unique in bearing a so-called C-terminal extension
(CTE) that protrudes from the globular structure [14]
Cleavage of eIF4GI by Lpro, both in vivo and in vitro,
is highly efficient [15–18] Nevertheless, the observed
Lpro proteinase concentration at which eIF4G is
cleaved during viral replication is much lower than
that required in vitro when purified recombinant
pro-teins are employed [16,18,19] For this reason, it has
been proposed that picornaviral proteinases activate
cellular proteinases, which cleave eIF4G in an indirect
reaction [20,21] However, such cellular proteinases
have as yet not been identified Ohlmann et al have
previously obtained evidence that the substrate for
Lpro is the eIF4GI–eIF4E complex [22] In addition,
for the HRV2 2A proteinase (2Apro), Haghihat et al
[23] demonstrated, using purified recombinant proteins,
that the eIF4GI–eIF4E complex was cleaved much
more efficiently than eIF4GI alone Pertinently, it has
been shown that yeast eIF4GI undergoes an
unfolded-to-folded transition on binding eIF4E [24,25] This
could be a reason why the eIF4GI–eIF4E complex is
the preferred substrate for picornaviral proteinases
Recently, we showed that FMDV Lpro and HRV2
2Apro indeed bind directly to the eIF4GI–eIF4E
com-plex, but much less well to eIF4GI alone [26]
Addi-tionally, we have shown that both of these proteinases
interact with their substrate eIF4GI at a site distant
from their cleavage site [26,27] For FMDV Lpro, we
minimized the binding site on eIF4GI to amino acids
640–669; in contrast, the enzyme cleaves eIF4GI
between residues G674 and R675 Here we show that
we can define this region further to the 13 amino acids
between residues 645–657 This region in eIF4GI
con-tains conserved basic residues, which are here
demon-strated to be involved in binding by Lpro The Lpro
binding domain for eIF4GI is located 25 A˚ from the
active site of the enzyme and comprises C133 as well
Results
Recently, we have shown that FMDV Lbprocan bind
to its substrate eIF4GI between amino acids 640–669 [26]; binding in the presence of eIF4E to eIF4GI frag-ments containing the eIF4E binding site was more efficient However, Lbpro cleaves between amino acids G674 and R675 We wished to further define this region between amino acids 640–669 and therefore cloned shorter fragments of eIF4GI, as shown in Fig 1A We started with an eIF4GI fragment contain-ing amino acids 260–657; RNA from this construct was translated in vitro in rabbit reticulocyte lysate
D C
Fig 1 Minimizing the eIF4GI binding domain of Lb pro (A) eIF4GI fragments used Sites for eIF4E binding and Lbprocleavage are indi-cated (B–D) 35 S-labelled proteins translated in vitro from the indica-ted cDNA fragments of eIF4GI (input lanes 1, corresponding to a quarter of that used in each pull-down) were incubated with GST (lanes 2) or GST-Lb pro C51A (lanes 3) Bound proteins were resolved
by SDS ⁄ PAGE and detected by fluorography All fragments were reproducibly synthesized as doublets, presumably due to initiation
of translation at two AUG initiating codons in close proximity.
Trang 3(RRL) in the presence of radiolabelled methionine.
The labelled protein containing eIF4GI residues 260–
657 was then incubated with the fusion protein GST–
LbproC51A, which had been expressed in bacteria and
purified by binding to glutathione-sepharose beads
The C51A mutation serves to inactivate the enzyme
during the pull-down assays Figure 1B shows that
the eIF4GI fragment 260–657 is bound by GST–
LbproC51A, but not by GST alone When we used an
eIF4GI fragment of amino acids 260–649 (Fig 1C),
binding to Lbprocould still be detected but was clearly
weaker in comparison to that containing amino acids
260–657 In contrast, a protein containing amino acids
260–645 (Fig 1D) was essentially not recognized by
the GST–LbproC51A fusion protein These results
demonstrate that the 13 amino acids in the region of
645–657 on eIF4GI are important for binding by
FMDV Lbpro Nevertheless, the presence of eIF4E
binding sequences on the eIF4G fragment and the
presence of eIF4E in the binding assay are required
for more efficient Lbprobinding
As a control, we examined the binding of
radiola-belled cortactin, a cellular protein that we have shown
to be cleaved in vitro by Lbpro but at much slower
rates than eIF4GI The radiolabelled cortactin is not
bound by the GST–LbproC51A fusion protein (data
not shown), indicating that the interaction of the
GST–LbproC51A complex protein with the eIF4GI is
specific and that it is probably responsible for the
rapid cleavage of Lbproobserved on eIF4GI
When we examined the 13 amino acids of eIF4GI
responsible for binding Lbpro as well as those
sur-rounding this sequence more closely, we found that the
sequence of eIF4GI comprised three basic residues (K643, K646 and R650), which were conserved between human eIF4GI and eIF4GII (Fig 2A) Ana-lysis of protein databases revealed that these residues were also present in eIF4GI sequences from several mammalian species such as sheep, cow, hamster, horse, mouse, pig and rabbit (EMBL accession numbers: AJ746218–AJ746224 inclusive) This conservation sug-gested that these basic residues might be recognized by residues in the Lbpro CTE and thus enable the inter-action to take place
To investigate this notion, we individually mutated these three residues to alanine in the plasmid con-taining the eIF4GI deletion 260–657 to give the cor-responding three plasmids shown in Fig 3A,B Radiolabelled proteins corresponding to the mutants were expressed in RRLs and their ability to be bound
by GST–LbproC51A in the pull-down assays was then examined The results are shown in Fig 3 and Table 1 The quantitation shows that about 12.5% of the input material is bound by the wild-type fusion protein in this assay The efficiency of the pull-down is probably limited by the presence of the GST part of the fusion protein, the small size of the radiolabelled fragment and the probability that a certain fraction of both binding partners are incorrectly folded
When we introduced the mutation K643A into the eIF4GI construct comprising residues 260–657, we found that binding by GST–LbproC51A was reduced
to between 80 and 90% of the wild-type level (Fig 3A, Table 1) However, the presence of the mutation K646A reduced binding by about 75% when com-pared to the wild type fragment (Fig 3A, Table 1)
Fig 2 (A) Sequence alignment of human eIF4G proteins The swissprot entries are I4G1_human (eIF4GI) and I4G3_human (eIF4GII) (B) Comparison of the C-terminal extensions of FMDV Lb pro , ERAV Lb pro and ERBV Lb pro Asterisks in (A) and (B) indicate conserved basic residues and acidic resi-dues, respectively Amino acids 183–195 in FMDV Lb pro were shown to be required for eIF4GI binding [26].
Trang 4Binding to the eIF4GI protein bearing the mutation
R650A was also reduced, but only by between 50 and
70% (Fig 3A,B, Table 1) The relatively low
involve-ment of K643 for binding was supported by the
behaviour of the double mutant K643AK646A
(Fig 3A,D); the binding is similar to that of the single
mutant K646A Finally, no binding to the triple
mutant K643AK646AR650A in which all three
con-served basic residues were substituted with alanine was
observed (Fig 3E)
The above results show that mutation of any of the
three conserved basic residues of eIF4GI impairs its
interaction with FMDV Lbpro but that the effects of
the mutations differ This implied that conserved acidic
residues should be present in Lbpro that represent the interaction partners of the basic eIF4GI residues We have shown previously that residues 183–195 of the
18 amino acid CTE as well as C133 of Lbpro were involved in the interaction of Lbpro and eIF4GI [28] Examination of the amino acids 183–195 of the CTE revealed two residues, D184 and E186 (Fig 2B), which are conserved in all seven serotypes, including those from Africa [29,30] ERAV Lpro is also responsible for cleavage of eIF4GI and eIF4GII [13] Analysis of the CTE of this enzyme revealed that both residues were also present in its CTE In contrast, in the CTE of equine rhinitis B virus (ERBV), Lpro appears not to cleave eIF4GI [13] Fittingly, there is a residue equival-ent to E186 in ERBV Lprobut not to D184 (Fig 2B)
We thus investigated the role of these amino acids in their interaction with eIF4GI by mutational analysis Figure 4A shows that mutations in the amino acid sequence of this region of Lbpro can be readily intro-duced by using an oligonucleotide cassette spanning the BsiWI and Bpu10I restriction sites In this way,
we generated the substitutions D184A, E186K and Q185RE186K, and investigated the ability of these
Lbpromutants to carry out self-processing and eIF4GI cleavage when expressed in RRLs Figure 4B (lanes 1–5) shows that self-processing of wild-type
Fig 3 Conserved basic residues in eIF4GI
are essential for Lb pro binding (A–E) 35 S
lab-elled proteins translated in vitro from the
indicated cDNA fragments of eIF4GI (input
lanes 1, corresponding to a quarter of that
used in each pull-down) were incubated
with GST (lanes 2) or GST-Lb pro C51A (lanes
3) Bound proteins were resolved by
SDS ⁄ PAGE and detected by fluorography.
Table 1 Efficiency of binding of mutated eIF4GI fragments to
GST–Lb pro C51A The % bound values are expressed relative to the
amount bound by the wild-type, which represents about 12.5% of
the total input Experiment 2 shows the quantitation of Fig 3.
eIF4GI fragment
% Bound Experiment 1 Experiment 2
Trang 5LbproVP4VP2 into Lbpro and VP4VP2 takes place
between 4 and 8 min after protein synthesis is initiated
Similar kinetics of self-processing were observed with
all three mutants tested (Fig 4B, lanes 6–27) To
examine eIF4GI cleavage by the newly synthesized
Lbpro, we took advantage of the presence of eIF4GI in
the RRLs and examined its fate during the synthesis
of Lbpro, as reported previously [15] Accordingly,
aliquots of the translation reactions were subjected to
SDS⁄ PAGE, the gels blotted onto poly(vinylidene
difluoride) membranes and probed with an antiserum
against the N-terminus of eIF4GI (Fig 4C, lower
pan-els) eIF4GI itself migrates as a series of bands with a
molecular mass of 220 kDa [31] The bands have
dif-ferent N-termini, which arise from the use of difdif-ferent
AUG codons during synthesis of eIF4GI [32,33]
Clea-vage of eIF4GI by Lbpro at its single recognition site
between G674R (numbering according to [32])
gener-ates a series of N-terminal cleavage products that are
detected by the N-terminal antiserum used here (cpN,
Fig 4C)
Figure 4C (lanes 1–5) shows that 50% of eIF4GI is
cleaved after 4 min with wild-type Lbpro However, the
mutation D184A (Fig 4C, lanes 6–12) showed a delay
in the cleavage of eIF4GI, the time-point of 50%
eIF4GI occurring only after 12–20 min Thus, we con-cluded that this residue is involved in eIF4GI recogni-tion The E186 mutant (lanes 13–19) also showed a similar delay in eIF4GI cleavage, with 50% eIF4GI cleavage being observed only after 12–20 min To investigate whether only the charges of the residues
184 and 186 were important for cleavage of eIF4GI or whether other residues in this region could exert an influence on the reaction, we constructed the double mutant LbproQ185RE186K and investigated its activity (lanes 20–26) Again, eIF4GI cleavage was impaired as 50% cleavage could only be seen after 20–30 min
To verify further that these residues were involved
in binding to eIF4GI, we expressed the three mutants
as GST-fusion proteins and examined their ability in pull-down assays to bind to eIF4GI In this case, we used the endogenous eIF4GI present in RRLs as a source of eIF4GI to ensure the best possible binding
to the GST–Lbprocomplexes In Fig 5A, the purity of the expressed GST fusion proteins can be seen; Fig 5B shows the results of the GST pull-down assays All mutant proteins showed weaker binding than that found in the wild-type (lane 2); furthermore, the extent
of binding correlated with the effect of these mutations
on eIF4GI The mutants GST–LbproC51AD184A and
Fig 4 Substitution of residues D184 and E186 in the CTE of FMDV Lb pro affect eIF4GI cleavage (A) Structure of the expression block of
Lb pro VP4VP2 showing the position of restriction sites used to introduce mutations into the CTE of Lb pro (B) Autoradiograms of proteins syn-thesized from the indicated RNAs Substituted residues are underlined Samples were taken at the times indicated and Lbproself-cleavage from VP4VP2 examined (marked with arrows) (C), Immunoblot with the samples from (B) probed with an anti-eIF4GI antiserum to monitor the eIF4GI cleavage by Lb pro ; intact eIF4GI and the cleavage products cpNare marked.
Trang 6GST–LbproC51AE186K (lanes 3 and 4, respectively)
showed significantly less binding to eIF4GI than
GST–LbproC51A (lane 2); with the mutant GST–
LbproC51AQ185RE186K, we observed only very weak
binding (lane 5) Similar results were also obtained
when the 35S-labelled eIF4GI fragment from 260 to
657 was employed (data not shown)
We showed in Foeger et al [26] that endogenous
eIF4GII in RRLs could also be bound by the GST–
Lbprocomplexes We therefore examined the ability of
the mutated GST–Lbprocomplexes to bind endogenous
eIF4GII Figure 5C (lane 2) confirms that the
wild-type GST–Lbpro complex binds the endogenous
eIF4-GII In contrast, binding of the mutant complexes to
eIF4GII is greatly reduced or almost undetectable
(Fig 5C, lanes 3–5) Furthermore, the binding of the
mutant complexes to endogenous eIF4GII mirrors that
to eIF4GI (Fig 5B,C) Thus, it seems likely that
resi-dues D184 and E186 are involved in recognizing both
eIF4GI and eF4GII
Taken together, these results strongly suggest that
there is a specific interaction between the residues
K643, K646 and R650 in eIF4GI, and the amino acids
picornaviral replication We have shown recently that this cleavage is mediated by regions distant from the active site of two different picornaviral proteinases, namely the papain-like Lbproof FMDV and the chym-otrypsin-like 2Apro of HRV2 [26,27] Furthermore, these regions interact on eIF4GI with amino acids that are not identical with the cleavage site of the particular enzyme
In this paper, we define the binding site recognized
by the Lbpro of FMDV to the 13 amino acids from residues 645–657 of eIF4GI This sequence is separated
by 17 amino acids from the cleavage site of Lbpro between residues 674 and 675 In contrast, the site on eIF4GI that is bound by HRV2 2Aprolies between resi-dues 600–674 [27], with the C–terminal boundary of this binding region only seven amino acids away from its cleavage site Furthermore, HRV2 2Aproonly binds
to eIF4GI when the binding site for eIF4GI is present and eIF4E is included in the binding assay Although these two parameters increase Lbpro binding, Lbpro is still capable of binding to eIF4GI fragments in their absence Thus, the two picornaviral proteinases recog-nize different sequences on eIF4GI; given their quite different structures, this is not unexpected
Investigation of the amino acids both within and adjacent to the region of eIF4GI to which FMDV
Lbpro binds revealed three basic amino acids which were conserved between human eIF4GI and eIF4GII,
as well as between the eIF4GI proteins of other mam-mals In addition, two acidic residues (D642 and D653) also appeared to be conserved in human eIF4GI and eIF4GII (Fig 2A) Furthermore, both of these aspartic acid residues are found in the seven animal eIF4GI sequences available in the database As it seemed possible that conserved charged residues might
be involved in the interaction, we examined the Lbpro CTE sequence from amino acids 183–195 These resi-dues had been shown previously to be important in the recognition of eIF4GI by Lbpro [26,28] In this sequence, we indeed noted a number of acidic and basic residues Close investigation of the sequences
of CTEs from other FMDV serotypes revealed that only residues D184 and E186 were present in all other serotypes, including the more distant South African
Fig 5 Specific mutations in the CTE of Lb pro reduce binding to
both eIF4GI and eIF4GII (A) Coomassie Brilliant Blue staining of
purified GST (lane 1) and modified GST-LbproC51A fusion proteins.
(B and C), 8 lL RRL (input lanes 0, corresponding to 2 lL of RRL)
were incubated with GST (lane 1) or the different GST-Lb pro C51A
fusion proteins (lanes 2–5) Bound eIF4GI (B) and eIF4GII (C) were
detected by immunoblotting using anti-eIF4GI and anti-eIF4GII sera.
Trang 7Territories (SAT) serotypes In addition, comparison
with the CTE sequence of ERAV Lpro, which has been
shown to be responsible for eIF4GI cleavage [13],
showed that D184 and E186 were also present in its
CTE In contrast, only E186 was present in the ERBV
Lpro sequence [34]; however, this enzyme appears not
to be responsible for eIF4GI cleavage [13] Thus, the
presence of D184 and E186 in the CTE of all Lpro
shown to be responsible for cleavage of eIF4G
pro-teins suggested to us that these residues might be
involved in interacting with the conserved basic
resi-dues of eIF4GI
To investigate this, we individually substituted the
three conserved basic residues in eIF4GI with alanine
residues Replacement of K646 reduced binding by
about 75% whereas replacement of R650 only reduced
binding by between 50 and 75% The replacement of
K643 had the least effect, with binding only being
reduced by 10–20% These results strongly suggested
an involvement of residues K646 and R650 in the
interaction with Lbpro This encouraged us to
investi-gate the role of amino acids D184 and E186 in the
cleavage of eIF4GI The substitution of D184 with
alanine or that of E186 with lysine both severely
delayed eIF4GI cleavage and led to a concomitant
decrease in binding of both eIF4GI and eIF4GII
Interestingly, the introduction of a second basic
resi-due (arginine in place of glutamine at 185) delayed
cleavage even further, suggesting that the overall
charge of this region is important for eIF4GI
recogni-tion
Figure 4 shows clearly, however, that eIF4GI
clea-vage still occurs when both D184 and E186 are
replaced by alanine One reason for this is the presence
of Lbpro residue C133 This residue is not part of the
CTE but lies close to it in the three-dimensional
struc-ture [14]; we showed previously that replacement of
this residue affects both the binding to and cleavage of
eIF4GI [26] However, the results here also do not rule
out further interactions between the CTE of Lbproand
the region 645–657 of eIF4GI involving other sequence
motifs or hydrophobic interactions not considered
here L188, found in the CTEs of FMDV and ERAV,
may be important in this respect
How can mutations in the amino acids 184–186
affect eIF4GI cleavage without affecting
self-process-ing? Examination of the structure of the Lbpro(Fig 6)
shows that residues D184 and E186 are at the opposite
side of the molecule from the active site and lie about
12 A˚ from C133, a residue which has also been shown
to be important for binding eIF4GI [26] Furthermore,
both residues protrude away from the globular domain
of the enzyme and do not appear to interact with any
residues in the globular domain or in the CTE Indeed, they are well positioned to interact with residues from another protein Thus, it seems that Lbpro, despite being one of the smallest papain-like enzymes, has been able to evolve a site which can significantly accel-erate cleavage of a host cell molecule without reducing the self-processing reaction Of the two reactions, the eIF4GI cleavage reaction appears to be more sensitive
to mutation than the self-processing reaction This emphasizes the importance of the interaction of Lbpro with the eIF4G proteins for the successful replication
of FMDV
In summary, we have defined closely the regions on eIF4GI and Lbpro that enable them to interact with each other A minimal binding site on eIF4GI between residues 645 and 657 has been identified, although binding is more efficient when eIF4G fragments con-tain the eIF4E binding site and eIF4E is present in the binding assay Once again, the versatility of viral pro-teins is amply illustrated Although viral propro-teins must remain small in order to limit genome size, they are still able to evolve domains away from the canonical active site which can interact with a second substrate and contribute to the efficiency of viral replication
Experimental procedures
Reagents
The FMDV Lpro is the most N-terminal protein on the FMDV polyprotein Lprofrees itself by cleavage between its own C-terminus and the N-terminus of VP4 As the initi-ation of protein synthesis on the FMDV RNA can occur at one of two AUG codons lying 84 nucleotides apart, two forms of Lpro(designated Labproand Lbpro) are synthesized
in the infected cell The reason for this is not clear, as both
Fig 6 Arrangement of Lb pro residues involved in recognizing eIF4GI Stereo diagram of Lb pro (green, a-helices; purple, b-sheets; yellow, coils) showing C133, D184, Q185 and E186 as balls-and-sticks The catalytic residues C51 (alanine in the crystal structure [14]) and H148 are also shown The drawing was produced using the program MOLSCRIPT [37,38] and rendered with RASTER 3 D [39] The PDB coordinates used for Lb pro were 1QOL, molecule G.
Trang 8font, Buckinghamshire, UK) as required [26] Fragments of
eIF4GI for in vitro translation were amplified from plasmid
pSKHC1, which contains the human eIF4GI cDNA from
amino acid 197–1600 [36], and cloned as EcoRI⁄ HincII
frag-ments into pBluescriptKS (Stratagene, La Jolla, CA, USA)
Mutations were introduced into the cDNAs for Lbproand
eIF4GI using standard PCR mutagenesis except for the
amino acid substitutions described in Fig 4 which were
introduced by replacing the 36 bp BsiWI and BpuI0I
frag-ment [28] of LbproVP4VP2 with the appropriate synthetic
oligonucleotides
The following antibodies were used Rabbit polyclonal
antiserum raised against the N-terminus of eIF4GI (kindly
provided by R Rhoads, Shreveport, LA, USA) was diluted
1 : 8000 Rabbit polyclonal antiserum raised against the
C-terminus of eIF4GII (kindly provided by N Sonenberg,
Montreal, Quebec, Canada) was diluted 1 : 2000
Secon-dary horse radish peroxidase (HRP)-conjugated antibodies
were diluted 1 : 10000 (BioRad, Hercules, CA, USA), and
second alkaline peroxidase (AP)-conjugated antibodies were
diluted 1 : 5000 (Sigma, St Louis, MO, USA)
Purification of GST fusion proteins
E coli JM101 cells were transformed with plasmids
enco-ding the GST-Lbpro fusion proteins or GST alone To
express GST-Lbpro, an overnight culture was diluted 1 : 10
in 50 mL medium, isopropyl thio-b-d-galactoside added to
a final concentration of 2 mm and the cells incubated at
30C for 3 h The proteins were purified on
glutathione-agarose resin (Amersham Biosciences) using standard
tech-niques
GST pull-down assays
Glutathione-sepharose beads coated with GST fusion
pro-teins were incubated in binding buffer (50 mm Tris⁄ HCl
pH 7.4, 10 mm EDTA, 150 mm NaCl) with either an
ali-quot (8 lL) of RRL or with radiolabelled in vitro translated
proteins for 2 h at 4C The amount of radiolabelled
pro-tein was adjusted so that the same amount was added in
each set of binding experiments After three washes with
binding buffer, bound proteins were eluted by boiling in
SDS⁄ PAGE loading buffer, resolved by SDS ⁄ PAGE and
visualized by western blotting and using the enhanced
chemiluminescence system (Pierce, Rockford, IL, USA) for
Transcription⁄ Translation system; Promega, Madison,
WI, USA) in the presence of [35S]methionine (20 lCi per reaction; Hartmann Analytic, Braunschweig, Germany) Labelled proteins were resolved by SDS⁄ PAGE and gels were dried and exposed to X-ray films In vitro translations
in RRLs (Promega) to examine Lbpro self-processing and eIF4GI cleavage were performed using in vitro transcribed RNAs as described previously [15,28]
Acknowledgements
This work was supported by the Austrian Science Foundation (grants P-16189 and P-17988) to T.S
We thank Bob Rhoads and Nahum Sonenberg for reagents
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