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Journal of Virology, September 2001, p. 7854-7863, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7854-7863.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Activity of the Hepatitis A Virus IRES Requires
Association between the Cap-Binding Translation Initiation Factor
(eIF4E) and eIF4G
Iraj K.
Ali,1
Linda
McKendrick,2
Simon J.
Morley,2 and
Richard J.
Jackson1,*
Department of Biochemistry, University of
Cambridge, Cambridge CB2 1GA,1 and
School of Biological Sciences, University of Sussex, Falmer,
Brighton BN1 9QG,2 United Kingdom
Received 16 January 2001/Accepted 29 May 2001
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ABSTRACT |
The question of whether translation initiation factor eIF4E and the
complete eIF4G polypeptide are required for initiation dependent on the
IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been
examined using in vitro translation in standard and eIF4G-depleted
rabbit reticulocyte lysates. In agreement with previous publications,
the HAV IRES is unique among all picornavirus IRESs in that it was
inhibited if translation initiation factor eIF4G was cleaved by
foot-and-mouth disease L-proteases. In addition, the HAV IRES was
inhibited by addition of eIF4E-binding protein 1, which binds tightly
to eIF4E and sequesters it, thus preventing its association with eIF4G.
The HAV IRES was also inhibited by addition of m7GpppG cap
analogue, irrespective of whether the RNA tested was capped or not.
Thus, initiation on the HAV IRES requires that eIF4E be associated with
eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied.
This suggests two alternative models: (i) initiation requires a direct
interaction between an internal site in the IRES and eIF4E/4G, an
interaction which involves the cap-binding pocket of eIF4E in addition
to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a
particular conformation which can be attained only if eIF4E is bound to
it, with the cap-binding pocket of the eIF4E unoccupied.
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INTRODUCTION |
It is now generally accepted that
picornavirus RNAs are translated by a mechanism of internal initiation,
in which the ribosome enters directly at an internal site within the
RNA rather than scanning from the physical 5' end (reviewed in
reference 2). The 5' untranslated region (UTR) of the
viral RNA has an IRES (internal ribosome entry site) about 450 nucleotides (nt) in length which is necessary and sufficient to promote
internal ribosome entry and internal initiation. On the basis of
primary and secondary structure conservation, the picornavirus IRESs
can be divided into one minor and two major groups: (i) hepatitis A
virus (HAV); (ii) entero- and rhinoviruses; and (iii) cardio-, aphtho-,
and parechoviruses. Internal initiation of translation on the IRESs of
the two major groups is thought to require all of the canonical initiation factors that are involved in the scanning mechanism except
that eIF4E is completely redundant and the requirement for eIF4G can be
fulfilled by just the C-terminal two-thirds fragment of this protein
(26, 27). Notably, the activity of these IRESs is not
inhibited (and may even be actually stimulated in certain circumstances) when eIF4G is cleaved by entero- or rhinovirus 2A
protease or foot-and-mouth disease virus (FMDV) L-protease (5, 6,
7, 28). These viral proteases cleave eIF4G to give (i) an
N-terminal one-third fragment which has the site for interaction of
eIF4G with eIF4E, the only translation initiation factor that binds
directly to 5' caps, and also a site for binding poly(A)-binding
protein; and (ii) a C-terminal two-thirds fragment which has two
distinct sites for interaction with eIF4A, the RNA helicase initiation
factor, and a site for binding eIF3 (14, 17, 18).
In sharp contrast to the two major types of picornavirus IRES, the
activity of the HAV IRES is strongly inhibited if eIF4G is cleaved by
the 2A protease or L-protease (4, 5, 7, 29). This implies
that the C-terminal two-thirds fragment of eIF4G, with its associated
eIF4A and eIF3, cannot support translation dependent on the HAV IRES.
However, it is not clear whether what is required is a larger fragment
of eIF4G, or whether the activity of this IRES actually needs eIF4E and
eIF4E-eIF4G association. Surprisingly, there appear to have been few
attempts, if any, to address these wider questions and to follow up the
initial finding of IRES inactivation by cleavage of eIF4G. In this
paper we remedy this deficiency by examining the effect on HAV IRES activity of eIF4E-binding protein 1 (4E-BP1), a protein which sequesters eIF4E tightly and as a consequence blocks its association with eIF4G, and of m7GpppG cap analogue, which binds to
eIF4E and prevents its interaction with the capped 5' ends of mRNAs
that are translated by the scanning mechanism. We show that internal
initiation on the HAV IRES has initiation factor requirements
remarkably similar to those for initiation of translation of capped
mRNAs by the scanning ribosome mechanism.
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MATERIALS AND METHODS |
Plasmid constructs.
The dicistronic construct with the HAV
IRES used in this work is pXLJ-HAV, described by Borman et al.
(5). It has the cDNA sequence of Xenopus laevis
cyclin B2 as the upstream cistron, followed by nt 44 to 378 of the HAV
sequence as intercistronic spacer, fused directly to the slightly
truncated form of the NS1 cDNA described by Borman and Jackson
(3), cloned into pGEM-2 (Promega) such that transcription
with T7 RNA polymerase generates sense RNA. With one exception, all of
the constructs with other viral IRESs are built on similar lines.
pXLJHRV10-611 has the complete human rhinovirus type 2 (HRV2) 5' UTR
(except the first 9 nt) fused to the same slightly truncated NS1 cDNA
sequence (3, 12). pXLPV1-747 has the complete 5' UTR and
the first 5 nt of coding sequence of poliovirus (PV) type 1 (Mahoney)
fused via a 34-nt linker to the full-length NS1 coding sequences
(12). The classical swine fever virus (CSFV) construct has
the first 423 nt of the CSFV (Alfort) sequence fused, via a 3-nt
linker, to the truncated NS1 sequences; the downstream cistron product is therefore the slightly truncated NS protein with an N-terminal extension consisting of the first 17 amino acids of CSFV coding sequence, plus one amino acid encoded by the 3-nt linker. All of the
above plasmids were linearized with EcoRI prior to in vitro transcription.
The upstream cistron was deleted from pXLJ-HAV to generate a
monocistronic derivative, pHAV-NS. pXLJ-HAV was cut with
HindIII and SalI, the ends were filled in,
and the plasmid was religated. pHAV-NS was likewise cut with
EcoRI prior to in vitro transcription.
The dicistronic construct with the encephalomyocarditis virus (EMCV)
IRES was generated from pEMCV L-VP0, which has been described
previously (
16). The cDNA encoding the slightly truncated
form
of NS1 and the NS1 3' UTR was excised from the previously
described
pJ0 (
3) by cutting with
SalI and
EcoRI, the overhanging ends
were filled in, and the fragment
was inserted into the blunted
EcoRI site of pEMCV L-VP0,
upstream of the EMCV IRES sequence.
A clone with the correct (sense)
orientation of the NS-related
cDNA sequence was selected. It was
linearized with
StuI prior
to in vitro
transcription.
Two constructs were used to generate monocistronic mRNAs that would be
translated by the scanning mechanism. One has the cDNA
sequences coding
for
unr (upstream of N-
ras) cloned into pET21d
(
13) and was linearized with
HindIII prior
to transcription.
The other (pXL4) codes for
X. laevis
cyclin A (
22) and was linearized
with
BamHI.
In vitro transcription and translation assays.
Transcription
of linearized plasmids by bacteriophage T7 RNA polymerase was carried
out exactly as described previously (8). In vitro
translation assays were carried out as described by Jackson and Hunt
(15). Briefly, the reactions consisted of 60 to 70% (by
volume) micrococcal nuclease-treated rabbit reticulocyte lysate and the
following additional components at the final concentrations stated: 100 or 70 mM KCl (as stated in the figure legends), 0.5 mM
MgCl2, 10 mM creatine phosphate, 50 µg of creatine
phosphokinase per ml, 15 µM hemin, 0.1 mM each amino acid except
methionine, 50 µg of calf liver tRNA (Boehringer) per ml, and 0.5 mCi
of [35S]methionine (SJ1515; Amersham Pharmacia Biotech)
per ml. Assays supplemented with m7GpppG or GpppG also
received additional MgCl2 at 0.8 mol/mol of cap analogue,
to counteract the chelating potential of the cap analogue; the ratio
was chosen empirically on the basis of the observed shift in
Mg2+ optimum caused by these cap analogues. In experiments
in which the HRV and PV IRESs were assayed, the reticulocyte lysate was replaced by a mixture (80:20, vol/vol) of reticulocyte lysate and HeLa
cell high-salt (HS)S100. The latter is essentially the complete
cytoplasmic extract minus salt-washed ribosomes; it is prepared by
making HeLa cell S10 postmitochondrial supernatant 0.5 M in KCl and 6 mM in magnesium acetate, centrifuging the ribosomes at
100,000 × g, and dialyzing the supernatant against
low-KCl buffer as described previously (12, 13).
Translation assays were incubated at 30°C for 60 min, and then the
translation products were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by
autoradiography. Hyperfilm 
max (Amersham Pharmacia Biotech) was used
for autoradiography, and quantitative densitometry of the films was
done using Phoretix software.
To generate FMDV L-protease by in vitro transcription and translation,
plasmid pMMl (
21) was linearized with
XbaI
prior
to in vitro transcription by T7 polymerase under conditions to
generate uncapped transcripts (
8). The RNA was translated
in
vitro at 50 µg/ml for 90 min under the conditions described above
except that the added KCl concentration was reduced to 50 mM.
The
reaction was then made 2 mM in CaCl
2, to reactivate the
micrococcal
nuclease, and left for 15 min at room temperature before
addition
of 5 mM EGTA. The protease preparation was diluted 100-fold
into
fresh lysate, which was then incubated for 10 min at 30°C to
effect
complete cleavage of the endogenous
eIF4G.
Recombinant L-protease and 4E-BP1 were expressed in
Escherichia
coli and purified as described previously (
23).
Rabbit reticulocyte
lysates were depleted of eIF4G by an affinity
column depletion
method described in detail elsewhere (
1);
for the experiments
described here, the lysates were made 70 mM in KCl
prior to addition
to the affinity matrix, rather than the 100 mM used
previously.
The expression in
E. coli and subsequent
purification of recombinant
p100 fragment of eIF4G was as described
elsewhere (
1).
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RESULTS |
Assay systems.
The standard approach in this work has
been in vitro translation of a dicistronic mRNA (with the HAV IRES as
intercistronic spacer) in rabbit reticulocyte lysates. The dicistronic
mRNA was that described by Borman et al. (5) and has an
upstream cistron coding for X. laevis cyclin B2 and a
downstream cistron coding for a slightly truncated version of the
influenza virus (A strain) NS1 protein. In view of the previous finding
that the optimum monovalent salt concentration for HAV IRES activity is
atypically low (5), the translation assays were carried
out with 70 mM added KCl rather than the 100 mM normally used for
translation of typical capped mRNAs. In several experiments,
comparisons were made between this dicistronic mRNA with the HAV IRES
and similar mRNAs with other picornavirus IRESs. When the comparison
was with the PV or HRV IRES, the reticulocyte lysate was replaced by a mixture of rabbit reticulocyte lysate and HeLa cell HS S100 (80:20, vol/vol) to provide the trans-acting factors which are
necessary for the activity of these two IRESs but which are either
absent from reticulocyte lysates or present only in very low abundance. We have not observed any major differences between the characteristics of HAV IRES function in this mixed system as opposed to the standard reticulocyte lysate system.
For some experiments, we used monocistronic mRNAs with the HAV IRES
linked to the slightly truncated NS1 reporter rather than
dicistronic
templates. The reason for this is that the HAV IRES
is rather weak, and
despite the use of a reduced KCl concentration
(70 mM), and even though
the RNA concentration was rather below
the saturating level, there were
indications that the apparent
activity of the HAV IRES was influenced
by the efficiency of the
competing translation of the upstream cistron
of the dicistronic
mRNA. For example, in a comparison of capped and
uncapped dicistronic
mRNAs, the IRES appears to be more active in the
latter case than
in the capped mRNA background, presumably because
there is much
more competition from upstream cistron translation when
the mRNA
is capped (see, for example, Fig.
4).
The HAV IRES is inhibited by recombinant FMDV L-protease and
L-protease expressed in vitro.
To verify that the HAV IRES behaves
in the same way in our system as reported previously by others
(4, 5), we first looked at the effect of recombinant
L-protease on translation of capped dicistronic mRNAs with the HAV,
HRV, or PV IRES. The results (Fig. 1)
show that, particularly at the two higher concentrations of protease,
there was inhibition of translation of the upstream (capped cistron)
and translation driven by the HAV IRES. In complete contrast,
translation dependent on the HRV and PV IRESs was significantly stimulated by L-protease, in agreement with previously published results (5). (The larger size of the downstream cistron
product translated from the PV IRES is because it has the full-length NS coding sequences, rather than a slightly truncated form, and this
sequence is fused to the initiation codon via a short linker sequence
rather than being linked directly to the initiation codon.)
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FIG. 1.
Recombinant FMDV L-protease inhibits HAV IRES activity.
Capped dicistronic mRNAs, each with the designated IRES, were
translated at 25 µg/ml in the mixed reticulocyte lysate-HeLa cell
HS-S100 system (see Materials and Methods), which had been preincubated
for 10 min at 30°C with the indicated concentration of recombinant
FMDV L-protease or with buffer (lanes C [control]). The concentration
of added KCl in the assays was 70 mM. Translation was for 60 min, and
the translation products were analyzed by SDS-PAGE followed by
autoradiography. The positions of the upstream (cyclin) cistron product
and downstream, IRES-dependent, NS-related product are shown. The
NS-related product translated from the PV IRES is larger because this
is full-length NS1 protein, rather than the slightly truncated form
linked to the HAV and HRV IRESs, and because the NS coding sequences
are joined to the authentic PV initiation codon via a short linker. The
yields of radiolabeled translation products of the upstream and
IRES-driven cistrons were determined by scanning densitometry and are
expressed relative to the yield in the corresponding control assay,
which was set at 100 (the underlined value).
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One possible reservation concerning these findings is that very high
concentrations of recombinant L-protease are needed to
effect the
cleavage of eIF4G in the reticulocyte lysate and to
obtain the effects
seen in Fig.
1. In contrast, L-protease expressed
by in vitro
translation appears to be at least 100-fold more active
(per microgram)
in eIF4G cleavage activity (
2). Accordingly,
we considered
it important to test whether these low levels of
in vitro-expressed
L-protease could also inhibit HAV IRES activity,
a question which does
not seem to have been addressed previously.
Figure
2 shows that, as expected, this
preparation of L-protease,
which caused complete cleavage of the
endogenous eIF4G (data not
shown), inhibited the translation of a
capped monocistronic RNA
(
unr mRNA) and the upstream cistron
of all capped dicistronic
mRNAs. In complete contrast, it stimulated
the translation of
uncapped monocistronic
unr mRNA, entirely
consistent with previously
published results (
22). Not
surprisingly, therefore, in the
case of the uncapped dicistronic mRNA
with the CSFV IRES, there
was also a stimulation of translation of the
upstream cistron,
which was very inefficiently translated not just
because the RNA
was uncapped but also because of strong competition by
the powerful
CSFV IRES (Fig.
2). As for IRES-dependent translation,
there was
no significant effect on the EMCV, CSFV, and PV IRESs, but
the
HRV IRES was quite strongly stimulated (Fig.
2). In complete
contrast,
however, the HAV IRES was strongly inhibited by this protease
preparation (Fig.
2), just as effectively as when much higher
levels of
recombinant L-protease were used (Fig.
1). (Here again,
the different
sizes of the IRES-dependent NS-related translation
product in Fig.
2
depend on whether it is the full-length or slightly
truncated form of
NS coding sequences that is present, and whether
these NS-related
sequences are fused directly to the initiation
codon or fused
indirectly, either via linker sequences as in the
case of the PV
construct or via viral coding sequences as for
the CSFV construct).
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FIG. 2.
The activity of the HAV IRES is inhibited by 4E-BP1 and
by FMDV L-protease expressed in vitro. All RNAs were translated at 25 µg/ml in the mixed reticulocyte lysate-HeLa cell HS-S100 system (see
Materials and Methods), which had been preincubated at 30°C as
follows: lanes a, 15-min preincubation with buffer (control); lanes b,
10-min preincubation with 4E-BP1 (10 µg/ml); lanes c, 5-min
preincubation with FMDV L-protease expressed in vitro; lanes d, 5-min
preincubation with FMDV L-protease followed by 10-min preincubation
with 4E-BP1 (10 µg/ml). The concentration of added KCl in all of the
assays was 70 mM. (A) All dicistronic mRNAs have an upstream cistron
coding for X. laevis cyclin B2 and a downstream cistron
coding for an influenza virus NS1 derivative, and all except for the
RNA with the CSFV IRES were capped. (B) Control assays carried out with
monocistronic mRNAs coding for unr in both capped and
uncapped forms. (C) The template was a capped dicistronic mRNA with a
slightly truncated form of the influenza virus NS1 coding sequence as
the upstream cistron, an EMCV IRES, and EMCV sequences coding for viral
L-VP0 as the downstream IRES-dependent cistron. In all cases,
translation was for 60 min and the translation products were analyzed
by SDS-PAGE followed by autoradiography. The positions of the upstream
cistron product and downstream, IRES-dependent product are shown. The
different sizes of the IRES-dependent NS-related translation product
are discussed in Results. The yields of radiolabeled translation
products of the IRES-driven cistrons (A and C) and of the single
product in the case of the monocistronic RNAs (B) were determined by
scanning densitometry and are expressed relative to the yield in the
corresponding control assay, which was set at 100 (the underlined
value).
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The HAV IRES is inhibited by addition of 4E-BP1, and the inhibition
is reversed by eIF4E.
Figure 2 also shows the results of
experiments in which the translation assays were supplemented with
4E-BP1, which binds to and sequesters eIF4E, preventing the interaction
of eIF4E with eIF4G to constitute the eIF4F holoenzyme complex
(25). Preincubation with 4E-BP1 inhibited the translation
of a capped monocistronic mRNA (coding for unr) and the
upstream cistron of all capped dicistronic mRNAs. At this
concentration, it had no marked or significant effect on translation of
uncapped monocistronic unr mRNA or on the EMCV, CSFV, HRV,
or PV IRES (Fig. 2). The HAV IRES was clearly the exceptional IRES in
that it was strongly inhibited (Fig. 2). Inhibition of the upstream
cistron of capped dicistronic mRNAs and of the HAV IRES activity could
be at least partially reversed by addition of eIF4E (Fig.
3), and the extent of this reversal was
dependent on the concentration of added eIF4E (I. K. Ali and R. J. Jackson, unpublished data).
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FIG. 3.
The inhibition of HAV IRES activity by 4E-BP1 can be
reversed by addition of eIF4E. Mixed reticulocyte lysate-HeLa cell
HS-S100 (see Materials and Methods) was preincubated for 10 min at
30°C with 4E-BP1 (10 µg/ml) or with buffer control (C); then KCl
was added to 70 mM together with the other components of the
translation assay, including capped dicistronic mRNAs, each with the
designated IRES, at 25 µg/ml. Where indicated, eIF4E (purified from
pig brain) was added at 25 µg/ml. Translation was for 60 min, and the
translation products were analyzed by SDS-PAGE followed by
autoradiography. The positions of the upstream (cyclin) cistron product
and downstream, IRES-dependent, NS-related product are shown. The
NS-related product translated from the PV IRES is larger because this
is full-length NS1 protein, rather than the slightly truncated form
linked to the HAV and HRV IRESs, and because the NS coding sequences
are joined to the authentic PV initiation codon via a short linker. The
yields of radiolabeled translation products of the upstream and
IRES-driven cistrons were determined by scanning densitometry and are
expressed relative to the yield in the corresponding control assay,
which was set at 100 (the underlined value).
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We also examined the effect of pretreating the system first with FMDV
L-protease and then with 4E-BP1, this sequence of pretreatments
being
dictated by the fact that cleavage of eIF4G by the protease
is known to
be inhibited by 4E-BP1, presumably because 4E-BP1
sequesters eIF4E and
thus strips the eIF4E from association with
eIF4G, which appears to
change the conformation of eIF4G to one
that it is uncleavable
(
11,
24). The results (Fig.
2) indicate
that with the
upstream citron of capped dicistronic mRNAs, the
effect of the protease
was epistatic to the influence of 4E-BP1.
This is consistent with the
fact, mentioned above, that cleavage
of eIF4G by the in vitro-expressed
protease was virtually complete.
If there had been significant amounts
of residual uncleaved eIF4G
driving the translation of these capped
mRNAs, addition of 4E-BP1
to the L-protease-treated lysate would have
been expected to cause
further inhibition of translation beyond that
due to the
protease.
The HAV IRES is inhibited by m7GpppG cap analogue but
not by GpppG.
Addition of m7GpppG cap analogue to
assays of dicistronic mRNAs caused the expected inhibition of
translation of the upstream cistron if the mRNA was capped but a
stimulation of translation of the 5'-proximal cistron if the RNA was
uncapped (Fig. 4). The latter effect,
which has been reported previously (9), may be the
consequence of relief of competition due to translation of short capped
fragments of globin mRNA which will be present in the micrococcal
nuclease-treated lysate but will not give rise to detectable
translation products. As for translation dependent on the HAV IRES,
this was inhibited by cap analogue, irrespective of whether the 5' end
of the dicistronic mRNA was capped or uncapped (Fig. 4).
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FIG. 4.
The activity of the HAV IRES is inhibited by
m7GpppG cap analogue. Capped or uncapped dicistronic mRNAs
with the HAV IRES were translated at 25 µg/ml in reticulocyte lysate,
in the presence of added KCl at 70 mM. Cap analogue
(m7GpppG) was added at 0 (lane C), 0.025, 0.05, 0.1, 0.2, and 0.4 mM, and additional MgCl2 was also added at 0.8 mol/mol of cap analogue. Translation was at 30°C for 60 min, and the
translation products were analyzed by SDS-PAGE followed by
autoradiography. The positions of the upstream (cyclin) cistron product
and downstream, IRES-dependent, NS-related product are shown. The
yields of radiolabeled translation products of the upstream and
IRES-driven cistrons were determined by scanning densitometry and are
expressed relative to the yield in the corresponding control assay,
which was set at 100 (the underlined value).
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At first sight it would appear that the HAV IRES is more sensitive than
the upstream capped cistron to inhibition by cap analogue
(Fig.
4).
However, this conclusion needs to be tempered by the
facts that capping
of in vitro transcripts is never 100% efficient
(in fact, it is only
about 70% efficient in our hands [
8])
and that
m
7GpppG actually stimulates translation of the upstream
cistron
of uncapped dicistronic mRNAs (Fig.
4). Thus, the observed
effect
of cap analogue on the upstream cistron of capped transcripts
in
fact represents the sum of two opposing effects: an inhibition
of
translation of the majority capped species, and a stimulation
of the
minority uncapped transcripts in the preparation. Thus,
the data will
underestimate the true sensitivity to inhibition
by cap analogue of
transcripts that are 100%
capped.
Another feature of the data of Fig.
4 which deserves comment is the
fact that although translation dependent on the HAV IRES
seems highly
sensitive to inhibition by m
7GpppG, there is a residual
amount, equivalent to about 25% of
the control, which appears to be
rather resistant to such inhibition.
There are two alternative types of
explanation which can be invoked
to account for this result. One
possibility is that there are
two distinct mechanisms of initiation
operating: the minority
of initiation events occurring via a mechanism
that is very resistant
to cap analogue, and the majority of initiation
occurring by another
route that is highly sensitive to such inhibition.
The alternative
explanation, if all initiation events follow the same
single pathway,
is that this mechanism must still be able to operate,
albeit at
somewhat lower efficiency, even when the cap-binding pocket
of
the eIF4E component of the eIF4F complex is occupied by cap
analogue.
This would be rather different from the effect of cap
analogue
on the translation of capped mRNAs: with natural mRNAs that
are
100% capped (as opposed to capped RNAs generated by in vitro
transcription,
which inevitably include some uncapped species), 0.4 mM
cap analogue
generally causes near-complete inhibition
(
1).
The data of Fig.
4 give the appearance that in the complete absence of
cap analogue, the HAV IRES was more active in the uncapped
dicistronic
mRNA background than if the mRNA was capped. We believe
that the
explanation for these differences lies in competition
between the
5'-proximal cistron and the IRES-dependent cistron.
The more efficient
translation of the first cistron in the capped
mRNA than the uncapped
version results, by competition, in a lower
IRES activity in the capped
than the uncapped mRNA
background.
In view of these complications caused by competition between the two
cistrons, we studied the effect of m
7GpppG cap analogue, or
a GpppG control, on the translation of
monocistronic mRNAs with the HAV
IRES. These monocistronic mRNAs
were generated by in vitro
transcription under conditions designed
to produce either uncapped,
m
7GpppG-capped, or GpppG-capped RNAs. The translation
assays show
that the HAV IRES in these monocistronic mRNAs was quite
strongly
inhibited by even the lowest concentration of
m
7GpppG (Fig.
5),
irrespective of whether the 5' end was uncapped,
GpppG capped, or
m
7GpppG capped.
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FIG. 5.
The inhibitory effect of m7GpppG cap
analogue on HAV IRES activity is independent of the nature of the 5'
end. Monocistronic mRNAs synthesized either as uncapped RNAs or with
GpppG or m7GpppG capped 5' ends, as indicated, were
translated at 20 µg/ml in rabbit reticulocyte lysate, in the presence
of added KCl at 70 mM. Cap analogues, either m7GpppG or
GpppG, as indicated, were added at 0 (lane C), 0.025, 0.05, 0.1 or 0.2 mM, and additional MgCl2 was also added at 0.8 mol/mol of
cap analogue. Translation was at 30°C for 60, min and the translation
products were analyzed by SDS-PAGE followed by autoradiography. The
yields of radiolabeled translation products were determined by scanning
densitometry and are expressed relative to the yield in the
corresponding control assay, which was set at 100 (the underlined
value).
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Ability of the C-terminal two-thirds of eIF4G to drive translation
dependent on the HAV IRES.
Entero and rhinovirus 2A proteases and
FMDV L-protease cleave the eIF4G component of the eIF4F holoenzyme
complex into an N-terminal one-third fragment, which has the binding
site for the cap-binding factor eIF4E, and a C-terminal two-thirds
fragment (hereafter designated p100), which interacts with eIF3 and
also has two binding sites for the eIF4A RNA helicase factor (14, 17, 18). Since these viruses shut off translation of capped host
cell mRNA, and as the proteases inhibit the translation of capped mRNAs
in vitro (4, 22, 23), it has been generally assumed that
p100 cannot support the translation of capped mRNAs. However, by the
use of a novel eIF4G depletion strategy we have recently shown that
capped mRNA translation can be driven by recombinant p100, provided
sufficient is added (1); our observations suggest that the
shutoff of host cell mRNA translation is due not to an intrinsic
inactivity of p100 but to a combination of limiting concentration and
affinity for capped mRNAs, coupled with strong competition by the viral
RNA for p100.
We have studied the activity of dicistronic mRNAs with the HAV IRES in
this eIF4G-depleted reticulocyte lysate system. As
we have shown
elsewhere, translation in this system is highly
dependent on add-back
of eIF4G derivatives (e.g., p100) but does
not require supplementation
with other initiation factors (
1).
eIF4G is essentially
completely (~95%) depleted, but depletion
of other factors is only
partial: eIF3, 10 to 20% depleted; eIF4A,
30 to 40% depleted; eIF4B,
10 to 20% depleted; and eIF4E, 20 to
30% depleted (
1).
When the dicistronic mRNAs with the HAV IRES were tested in the
depleted system, the translation of both cistrons was, not
surprisingly, impaired (Fig.
6). Addition
of p100 restored the
translation of both cistrons, and this rescue was
completely resistant
to inhibition by m
7GpppG cap analogue
in the case of both the upstream scanning-dependent
cistron and the HAV
IRES-dependent cistron (Fig.
6). There was
also an increase in the
yield of incomplete products of translation
of the upstream cistron.
However, it should be noted that in general
the same set of incomplete
products was also seen, albeit in lower
yield, in the control
translation assays in nondepleted lysate.
In these controls, the
addition of m
7GpppG cap analogue affected the yield of
incomplete products and
of the major product in the same way: both
types of product were
inhibited in the case of the capped transcript
but stimulated
in the case of the uncapped species. In other words, the
incomplete
products must have been initiated by the same mechanism as
the
major product. Taken together with other criteria described
previously
(
8), this implies that most of the incomplete
products arose
from premature termination of translation initiated at
the correct
site, rather than illegitimate initiation at internal
sites.
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|
FIG. 6.
Translation driven by the HAV IRES can be supported by
the p100 fragment (C-terminal two-thirds) of eIF4G. Capped and uncapped
dicistronic mRNAs, as indicated, were translated at 25 µg/ml in
either eIF4G-depleted reticulocyte lysate or parent (nondepleted
lysate), in the presence of added KCl at 70 mM. Cap analogues, either
m7GpppG or GpppG, were added at 0.4 mM, where indicated,
together with 0.32 mM additional MgCl2. In lanes labeled
"p,", recombinant p100 was added at 20 µg/ml. Translation was at
30°C for 60 min, and the translation products were analyzed by
SDS-PAGE followed by autoradiography. The positions of the upstream
(cyclin) cistron product and downstream, IRES-dependent, NS-related
product are shown. The yields of radiolabeled translation products of
the upstream and IRES-driven cistrons were determined by scanning
densitometry and are expressed relative to the yield in the
corresponding control assay, which was set at 100 (the underlined
value).
|
|
It can be seen that the rescue of upstream cistron translation was
quite significantly more efficient than restoration of
IRES-dependent
translation (Fig.
6). This contrasts with what
is observed when
dicistronic mRNAs with the EMCV or Theiler's
murine encephalomyelitis
virus (TMEV) IRES are studied in the
same system, when it is invariably
the case that IRES-dependent
cistron translation is rescued very
efficiently, but translation
of the upstream cistron is rather
inefficient (
1). These observations
suggest that the HAV
IRES competes rather poorly against scanning-dependent
mRNAs or
cistrons for p100, but that p100 interacts preferentially
with the EMCV
and TMEV IRESs as opposed to scanning-dependent
mRNAs. Thus,
the hierarchy of the functional interactions of p100
with mRNAs is EMCV
or TMEV IRES
scanning-dependent mRNAs > HAV
IRES.
Because of the complication of competition between the two cistrons of
the dicistronic mRNA, the experiments with eIF4G-depleted
lysate were
repeated using a monocistronic mRNA with the HAV IRES.
The results show
that in the absence of any competition from translation
of another
cistron, addition of high concentrations of p100 stimulated
the HAV
IRES activity almost as effectively as the translation
of an uncapped
mRNA initiated by the conventional scanning mechanism
(Fig.
7). Moreover, the dependence of rescue on
p100 concentration
was not very different for an mRNA with the HAV IRES
than for
scanning-dependent capped or uncapped mRNAs (Fig.
7). To put
these
dose-response assays into a physiological perspective, our
previous
results indicate a concentration of endogenous eIF4F in the
lysate
equivalent to ~3.0 µg of p100 per ml (
1).
View larger version (38K):
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|
FIG. 7.
Dose response of p100 rescue of HAV IRES activity.
Monocistronic XL4 mRNA (coding for X. laevis cyclin A), in
either capped or uncapped form, and uncapped monocistronic HAV-NS RNA
were translated at 20 µg/ml either in the parent (nondepleted) lysate
(lanes C) or in eIF4G-depleted lysate supplemented with the designated
concentrations of recombinant p100. Translation was at 30°C for 60 min, and the concentration of added KCl was either 70 mM (both uncapped
RNAs) or 100 mM (capped XL4 mRNA), as indicated. The translation
products were analyzed by SDS-PAGE followed by autoradiography. The
yields of radiolabeled translation products were determined by scanning
densitometry and are expressed relative to the yield in the
corresponding control assay, which was set at 100 (the underlined
value).
|
|
In the case of capped mRNAs translated by the scanning mechanism, we
have previously shown that addition of p100 to control,
(nondepleted)
lysate has very little influence apart from a slight
increase in the
yield of products from those mRNAs which are poorly
translated
(
1). Essentially the same result was seen in the
case of
translation dependent on the HAV IRES. When the uncapped
dicistronic
mRNA was translated in the standard, nondepleted lysate,
addition of
p100 stimulated translation of the upstream cistron,
in agreement with
previously published results (
9), but had
no effect on
IRES-dependent translation (Ali and Jackson, unpublished).
With the
capped dicistronic mRNA, addition of p100 stimulated
upstream cistron
translation only marginally but increased the
yield of IRES-dependent
product until it became equal to the yield
obtained from the uncapped
version of the dicistronic
template.
We have also previously shown that addition of p100 can reverse the
inhibition of translation of capped mRNAs in standard
lysate caused by
addition of either FMDV L-protease, m
7GpppG cap analogue,
or 4E-BP1 (
1). Here again, p100 also effected
a remarkably
similar rescue if HAV IRES activity had been inhibited
by one of these
agents in a nondepleted standard lysate system
(Ali and Jackson,
unpublished).
| |
DISCUSSION |
It is widely recognized that the HAV IRES differs from all other
picornavirus IRESs in that it is rendered inactive if the endogenous
eIF4G is cleaved by entero- or rhinovirus 2A protease or FMDV
L-protease (4, 5, 7, 29). We have shown here that it is
also inhibited by m7GpppG cap analogue and by 4E-BP1, which
binds to and sequesters eIF4E, preventing its association with eIF4G to
generate the complete eIF4F complex (25). This is also in
complete contrast to the other picornavirus IRESs which are not
inhibited by 4E-BP1 (Fig. 2) or by cap analogue (Ali and Jackson,
unpublished). In its sensitivity to inhibition by all three reagents
(protease, cap analogue, and 4E-BP1), translation dependent on the HAV
IRES resembles the translation of capped mRNAs by the scanning ribosome
mechanism. This similarity also extends to the fact that
supplementation of the eIF4G-depleted lysate with p100 in sufficient
concentrations can support both scanning-dependent translation and
initiation on the HAV IRES (Fig. 6 and 7).
A reflection of the close parallel between translation initiation on
capped mRNAs and on the HAV IRES is that the translation of both
cistrons of a capped dicistronic mRNA with the HAV IRES responds in the
same way to protease or 4E-BP1 (Fig. 2) or cap analogue (Fig. 4). This
is of some concern because it raises the issue of whether the
translation of the downstream cistron really is via an internal
initiation mechanism in the normally understood meaning of that term.
Is it possible, for example, that the only interaction between eIF4F
and the capped dicistronic mRNA is at the 5' cap (via interaction of
the eIF4E subunit with the cap) and that the downstream cistron is
translated by the eIF4F "reaching over" to deliver the 40S
ribosomal subunit to the intercistronic IRES, which would require a
looping out of the whole upstream cistron? A mechanism whereby eIF4F
bound solely at the 5' cap, sometimes delivering the 40S subunits to a
cap-proximal site prior to scanning and sometimes delivering the
subunits to the IRES, would explain why the translation of both
cistrons responds in parallel to agents that perturb eIF4F-cap
interactions. It is a matter of semantics whether such a hypothetical
mechanism should be classified as internal initiation or
pseudo-internal initiation, but it would certainly be quite different
from what is believed to be the mechanism of internal initiation of
translation of other picornavirus RNAs.
Arguing against such a hypothetical model, there are a number of
situations in which the translation of the IRES-dependent cistron does
not parallel the translation of the upstream cistron. For example, when
uncapped and capped dicistronic mRNAs are compared, the upstream
cistron product is synthesized less efficiently from the uncapped
species than from the capped mRNA, yet the IRES-dependent cistron
translation is more efficient when the mRNA is uncapped rather than
capped (Fig. 4). Any interaction of eIF4F with the 5' end of the
uncapped mRNA is likely to be different in nature from its interaction
with a 5' cap, but this difference clearly does not affect the
translation of the upstream and downstream cistrons in the same way.
Moreover, when m7GpppG is added to translation assays of
uncapped dicistronic mRNA, it inhibits IRES-dependent cistron
translation but actually stimulates upstream cistron translation (Fig.
4), possibly through relief of the competitive influence of translation
of capped fragments of globin mRNA. Finally, it is pertinent that
translation of monocistronic mRNAs with the HAV IRES was equally
sensitive to inhibition by m7GpppG cap analogue, regardless
of the chemical identity of the 5' end of the RNA, yet the nature of
the interaction of eIF4F with the mRNA is strongly influenced by the
nature of the 5' end. All of these observations argue against the
notion that the 40S ribosomal subunit is delivered to the initiation
codon of the downstream (IRES-dependent) cistron by an eIF4F complex
that is bound at the very 5' end of the mRNA, whether dicistronic or monocistronic.
These considerations suggest that translation driven by the HAV IRES is
dependent on some functional relationship between eIF4F and the IRES
itself, quite likely a direct physical interaction between eIF4F and
the IRES, rather than an interaction of eIF4F with the 5' end itself.
Nevertheless, it is clear that the function of eIF4F in supporting
initiation on the HAV IRES is absolutely dependent on the presence of
eIF4E in the eIF4F complex, since it is inhibited by 4E-BP1 (Fig. 2 and
3), which in effect strips eIF4E from the eIF4F complex but does not
inhibit the ability of the eIF4E to interact with 5' caps
(25); it is further absolutely dependent on the
cap-binding pocket (19) of this eIF4E, since it is
inhibited by m7GpppG cap analogue. Two alternative models
can be advanced to account for these findings. One invokes a direct
interaction between the eIF4E component of eIF4F with a specific
internal site in the HAV IRES, an interaction which would necessarily
involve the cap-binding pocket of eIF4E in order to explain inhibition
of the IRES by m7GpppG cap analogue. In this model, the
postulated site-specific eIF4E-IRES interaction would, together with
perhaps some direct eIF4G-IRES interactions (10), position
the eIF4G component of the eIF4F complex in the appropriate proximity
and orientation to deliver the 40S ribosomal subunit to the 3' end of
the HAV IRES, at or very close to the initiation codon. The principal strength of this model is that it provides a ready explanation for
inhibition of the IRES by m7GpppG cap analogue, but the
main problem with it is that the crystal structure of eIF4E-cap
analogue complex (19) makes it hard to see how the
cap-binding pocket of eIF4E could interact with RNA at an internal site.
The alternative model, which is advanced in the accompanying paper,
(7a), is that HAV IRES activity requires the eIF4F
complex, complete with associated eIF4E, not because the eIF4E
component actually interacts with the IRES but because the eIF4E
subunit must be associated with eIF4G in order for the eIF4G to be able to adopt a conformation suitable for direct interaction with the IRES,
presumably at a specific site. One advantage of this model is that it
does not require an interaction of the cap-binding pocket of the eIF4E
component of eIF4F with an internal site in the IRES. Another is that
it is indeed quite well established that the withdrawal of eIF4E from
the eIF4F complex as a consequence of 4E-BP1-eIF4E interaction does
cause a significant change in the conformation of the eIF4G, such that
it now cannot be cleaved by picornavirus proteases (11,
24). In addition, structural studies of a peptide representing
the site on eIF4G which interacts with eIF4E have shown that this
peptide undergoes a considerable conformational change when it binds to
eIF4E (20).
On the other hand, this model has difficulty explaining inhibition of
the HAV IRES by m7GpppG cap analogue. It needs to postulate
that the binding of cap analogue to the cap-binding pocket of eIF4E in
the eIF4F complex results in a conformational change in the associated
eIF4G. The crystal structure of the eIF4E-m7GDP complex
shows that the protein resembles a cupped hand: the cap
analogue-binding pocket is on the concave side, and it is the other
side, the convex or dorsal face, which interacts with eIF4G (19,
20). Thus, a model which posits that interaction of the eIF4E-4G
complex with cap analogue causes a conformational change in the eIF4G
moiety would seem to require that the binding of cap analogue to the
concave surface of eIF4E induce a conformational change in the
diametrically opposite (convex) face, which in turn induces a
conformational change in the associated eIF4G. On present evidence this
seems improbable. Admittedly we do not know the structure of eIF4E per
se, only the crystal structure of the eIF4E-m7GDP complex
(19), and it would be fair to say that until we have the
difference map between these two states of eIF4E, we cannot
definitively rule out the possibility that binding of cap analogue
causes a change in the conformation of the opposite (convex) face of
eIF4E. However, we can say that if cap analogue binding to the eIF4E
moiety does induce a change in the conformation of eIF4G, it must be a
conformational change very considerably more subtle than that caused by
withdrawal of eIF4E from the complex by interaction with 4E-BP1, since
we find that although addition of 4E-BP1 to a lysate prevents cleavage
of the endogenous eIF4G by L-protease, as already reported previously
by others (11, 24), addition of m7GpppG cap
analogue has absolutely no influence on the rate or efficiency of eIF4G
cleavage by the protease (Ali and Jackson, unpublished).
In conclusion, we have extended previous work showing that the HAV IRES
is inhibited by cleavage of eIF4G by viral proteases (4, 5, 7,
29) to demonstrate that initiation on this IRES is unique among
picornavirus IRESs in exhibiting a strong requirement for eIF4E,
specifically eIF4E in association with eIF4G. We are left with two
alternative models to explain this surprising eIF4E requirement, but
current technology cannot at present distinguish between these two explanations.
These results raise interesting questions concerning the evolution of
picornaviruses. Current hypothesis envisages that all modern day
picornaviruses evolved from a single common ancestral virus. Did the
most recent common ancestor have initiation factor requirements
resembling those of HAV? Or did it more closely resemble EMCV in having
simpler requirements, and the branch which evolved to modern day HAV
(re)acquired the need for eIF4E in association with eIF4G? It seems
much more likely that the most recent common ancestor had factor
requirements similar to those of HAV, and that the requirement for
eIF4E and the N-terminal part of eIF4G was lost in all branches of the
evolutionary tree except that which has given rise to modern-day HAV.
| |
ACKNOWLEDGMENTS |
We thank Andy Borman and Kathie Kean for the gift of pXLJ-HAV;
Deirdre Scadden for the gift of pig brain eIF4E; and Simon Fletcher,
Ann Kaminski, Sarah Hunt, and Nancy Standart for other constructs. We
also thank C. U. T. Hellen (SUNY Health Center, Brooklyn,
N.Y.) and J. Lawrence, Jr. (University of Virginia) for providing
reagents and Rosemary Farrell for providing technical assistance and
infrastructure support to the R.J.J. group.
This work was supported by grants from the Wellcome Trust to R.J.J.
(051424) and S.J.M. (040800, 045619, 056778, 057494, and 058915).
S.J.M. is a Senior Research Fellow of the Wellcome Trust, and I.K.A.
was supported by a Medical Research Council postgraduate research studentship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom. Phone: (44) 1223-333682. Fax: (44) 1223- 766002. E-mail: rjj{at}mole.bio.cam.ac.uk.
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Journal of Virology, September 2001, p. 7854-7863, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7854-7863.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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