(Received for publication, April 20, 1995)
From the
Cap-dependent binding of mRNA to the 40 S ribosomal subunit
during translational initiation requires the association of eukaryotic
initiation factor 4G (eIF4G; formerly eIF-4 and p220) with other
initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells
by picornaviruses results in proteolytic cleavage of eIF4G and
generation of a cap-independent translational state. Rhinovirus 2A
protease and foot-and-mouth-disease virus L protease were used to
analyze the association of eIF4G with eIF4A, eIF4E, and eIF3. Both
proteases bisect eIF4G into N- and C-terminal fragments termed cp
and cp
. cp
was shown to contain the
eIF4E-binding site, as judged by retention on
m
GTP-Sepharose, whereas cp
was bound to eIF3
and eIF4A, based on ultracentrifugal co-sedimentation. Further
proteolysis of cp
by L protease produced an 18-kDa
polypeptide termed cp
which retained eIF4E binding
activity and corresponded to amino acid residues 319-479 of
rabbit eIF4G. Further proteolysis of cp
yielded several
smaller fragments. cp
(
887-1402) contained the
eIF4A binding site, whereas cp
(
480-886)
contained the eIF3 binding site. These results suggest that cleavage by
picornaviral proteases at residues 479-486 separates eIF4G into
two domains, one required for recruiting capped mRNAs and one for
attaching mRNA to the ribosome and directing helicase activity. Only
the latter would appear to be necessary for internal initiation of
picornaviral RNAs.
Translation of eukaryotic cellular mRNA into protein is a
complex process involving nearly 200 RNA and protein components
interacting in a regulated fashion to ensure timely expression of
genetic information(1) . Viruses often alter the host cell
translational machinery to allow more efficient expression of virally
encoded proteins. One of the most dramatic examples of this occurs upon
picornaviral infection. The picornaviridae are small, icosohedral,
positive-stranded RNA viruses of considerable clinical and veterinary
importance, containing such members as poliovirus, rhinovirus,
coxsackievirus, encephalomyocarditis virus, and foot-and-mouth-disease
virus (FMDV)()(2, 3) . Infection of
mammalian cells by most members of this family (all genera except
cardioviruses and hepatitis A virus) impairs the ability of the host
cell to translate capped mRNAs. Translation of the uncapped viral RNA
still proceeds, however, by a cap-independent mechanism whereby
ribosomes bind internally at specific sites on the viral
RNAs(4, 5) . Thus, picornaviral infection results in
the conversion of the predominant mode of translation initiation from
cap-dependent to capindependent.
The host cell shutoff by
rhinoviruses, enteroviruses, and aphthoviruses is thought to be
mediated, at least in part, by the virally induced cleavage of a
protein synthesis initiation factor,
eIF4G()(6, 7, 8) . The loss of the
ability to translate capped host mRNAs correlates with the conversion
of eIF4G, detected immunologically, from a cluster of bands migrating
on SDS-PAGE at 200-220 kDa to one of 100-130
kDa(6, 9) . This event is mediated by virally encoded
proteases(10, 11) . The cleavage products of eIF4G
detected in vivo are electrophoretically identical to those
generated in vitro using highly purified recombinant forms of
the viral proteases(12) . Although early evidence suggested
that viral proteases played only an indirect role in eIF4G
proteolysis(13, 14) , recent results support direct
cleavage of eIF4G by virally encoded
proteases(15, 16, 17) . However, the
observation that eIF4G cleavage is not sufficient for total host cell
shutoff in vivo(18, 19) suggests that
additional events may play a role in the inhibition of cap-dependent
translation.
eIF4G functions during initiation of translation
through association with other members of the eIF4 group of translation
factors (reviewed in (20, 21, 22) ). This
group includes eIF4A, a 46-kDa bi-directional ATP-dependent helicase;
eIF4B, a 70-kDa RNA-binding phosphoprotein which enhances eIF4A
activity; eIF4E, a 25-kDa cap-binding phosphoprotein; and eIF4G itself,
a 154-kDa phosphoprotein which can be isolated in complexes with eIF3,
eIF4A, eIF4B, and eIF4E (23, 24, 25) . The
best characterized of these complexes, eIF4F, consists of eIF4A, eIF4E,
and eIF4G(23) . These polypeptides collectively recognize the
mGTP-containing cap, unwind mRNA secondary structure, and
facilitate binding of the 40 S ribosomal subunit.
The mechanism by which cleavage of eIF4G by picornaviral proteases contributes to an inhibition of cap-dependent translation is not understood, but some functions associated with eIF4F appear to be inactivated. Following poliovirus infection, cross-linking of eIF4 polypeptides to the cap structure is altered(9, 26, 27) . The pattern of cap-binding protein complexes recovered from infected cells is distinctly different from that of uninfected cells, suggesting that infection disrupts macromolecular complexes important for cap-dependent initiation(28, 29) . Since no alteration in other eIF3 or eIF4 polypeptides is apparent as a result of infection (6, 30, 31) , cleavage of eIF4G is thought to be responsible for the observed changes. This is supported by the fact that protein complexes containing intact eIF4G restore cap-dependent translation in extracts of infected cells(23, 32, 33) . Also, extracts of uninfected cells, when treated with purified viral proteases, lose their ability to translate capped mRNA, but addition of eIF4G-containing complexes restores this activity(8, 16) .
Not only does cleavage of eIF4G result in inactivation of some functions important for cap recognition, but there is also evidence that eIF4G may be important for cap-independent translation of viral RNAs. Addition of the eIF4F complex stimulates in vitro translation from picornaviral internal ribosome entry site sequences (34, 35, 36) . In contrast to cap-dependent initiation, cleavage of eIF4G by picornaviral proteases does not abrogate this stimulation but rather enhances it(16, 37, 38) . This suggests that eIF4G cleavage products play a direct role in cap-independent translation of viral RNAs(16, 37) .
Picornaviral proteases which
cleave eIF4G fall into two separate classes. The 2A proteases of rhino-
and enteroviruses are small thiol proteases with structural
similarities to chymotrypsin and -lytic
protease(39, 40, 41) . The primary cleavage
site in rabbit eIF4G is Arg
-
Gly
(15) , although a secondary site of unknown
location has been suggested for the 2A protease of HRV2 (12) .
The L protease of FMDV more closely resembles papain in structure (42) . It initially cleaves eIF4G at
Gly
-Arg
and subsequently at multiple sites
as yet unidentified(12) .
To understand better the role of eIF4G in cap-dependent and -independent initiation, we have examined the effect of proteolysis on the association of eIF4G with other initiation factors using recombinant viral proteases. We provide evidence that proteolysis of eIF4G at the primary site separates functional domains. We also report the localization of a second L protease cleavage site in eIF4G which further defines the binding region for eIF4E. These results suggest a model for the role of eIF4G cleavage products in translation of picornaviral RNA.
Figure 1:
Time course of cleavage
of eIF4G by L protease of FMDV. eIF4F was digested with recombinant L
protease in the absence (lanes 1-7) or presence (lanes 8-14) of eIF3 as described under
``Experimental Procedures.'' Aliquots were removed at the
indicated times and subjected to SDS-PAGE on 10% gels. A,
silver-stained gel. B, immunoblot probed with
anti-eIF4G antibodies. C, immunoblot
probed with anti-eIF4G
antibodies. The
positions of standard proteins of the designated molecular masses
(
10
) are indicated on the left. The
positions of uncleaved eIF4G, the various cleavage products, eIF4A, and
eIF4E are indicated on the right.
Figure 2: Summary of viral protease cleavage of eIF4G. Long rectangles indicate the 1402-amino acid residue rabbit eIF4G molecule or various cleavage products (proportional in length to the molecular masses of the fragments). Precise cleavage sites for 2A and L proteases in rabbit eIF4G are indicated by vertical lines through amino acid sequences. Approximate cleavage sites are indicated by lines through the rectangles. The locations in eIF4G of synthetic peptides used to generate antibodies are shown at the top with inclusive amino acid residue numbers (referring to the human sequence).
Further
digestion with L protease caused the cp fragments to
disappear and a heterogeneous cluster of fragments, designated
cp
, to appear (A, lanes 4-7). All members
of the cp
cluster arose simultaneously and therefore are
likely to have resulted from cleavage of cp
at a single
site. The heterogeneity of cp
is similar to that of
cp
and eIF4G itself, suggesting that the source of
heterogeneity in eIF4G is localized to the cp
region.
Immunoblotting with an anti-eIF4G
antibody
detected a band with estimated molecular mass of 28 kDa, designated
cp
, which appeared simultaneously with the degradation of
cp
(B, lanes 2-4). With further digestion,
some of cp
was converted to smaller species termed
cp
(B, lanes 4-7). Immunoblotting with
anti-eIF4G
antibodies (B) and
anti-eIF4G
antibodies (data not shown)
indicated that cp
and cp
contained amino acid
residues 327-416, whereas cp
did not. This permitted
the orientation of cp
, cp
, and cp
as shown in Fig. 2.
Further cleavage of the 103-kDa
cp yielded a number of smaller fragments
(cp
-cp
). The relative abundance and
kinetics of appearance of the fragments together with immunoblotting
with anti-eIF4G
(Fig. 1C) and
anti-eIF4G
(12) antibodies is
consistent with the following order of events. Initial cleavage of
cp
by L protease can occur at one of two nearby sites (see Fig. 2). This yields two overlapping polypeptides, cp
(59 kDa) and cp
(55 kDa), both of which can be seen
in A, lanes 4-7, and both of which react with
anti-eIF4G
antibodies(12) . Secondary
cleavage of cp
generates cp
(note gradual
loss of cp
and appearance of cp
in Fig. 1A). When cp
is initially cleaved to
cp
, the N-terminal half is represented by cp
(40 kDa), which reacts with anti-eIF4G
antibodies (C, lanes 3-7, and Fig. 2). When
cp
is initially cleaved to cp
, the N-terminal
half is represented by cp
(45 kDa), which also reacts with
anti-eIF4G
antibodies (C, lanes
3-7). With longer times of incubation, cp
and
cp
disappear simultaneous with the appearance of cp
(35 kDa; C, lanes 4-7). Neither cp
,
cp
, nor cp
were recognized by
anti-eIF4G
(data not shown). These results
indicate that cp
and cp
are derived from the
C-terminal portion of cp
and that cp
,
cp
, and cp
are derived from the N terminus (Fig. 2). Based on the SDS-PAGE-derived molecular masses,
cp
and cp
approximately correspond to amino
acid residues 480-886 and 887-1402 of rabbit eIF4G, respectively.
eIF3 is a multisubunit initiation factor that associates with eIF4F (29) . Previously we showed that eIF3 does not affect cleavage
of eIF4G by 2A proteases from HRV2 and coxsackievirus serotype
B4(15) . However, Wyckoff et al.(49, 50) have presented evidence that eIF3 is
required for cleavage of eIF4G by poliovirus 2A protease. To determine
if eIF3 affected either the rate or sites of cleavage of eIF4G by FMDV
L protease, we repeated the time course in the presence of this factor
at a concentration equimolar to that of eIF4F (Fig. 1, lanes
8-14). No cleavage of eIF4E or the various subunits of eIF3
was observed. The rate of appearance of the eIF4G cleavage products was
unchanged except for a slight heterogeneity of the cp band; eIF3 caused a reduction in cp
and the
appearance of a new band
1 kDa smaller in size (cf.lanes 4-7 with lanes 11-14 in C). This suggests that eIF3 may block the accessibility of a
protease site or expose a new one in this region of eIF4G.
Figure 3:
mGTP-Sepharose column
fractionation of eIF4F treated with HRV2 protease 2A. eIF4F was
incubated with 2A protease and fractionated on
m
GTP-Sepharose as described under ``Experimental
Procedures.'' Aliquots of fractions were subjected to SDS-PAGE on
an 8.5% gel. A, Coomassie Blue-stained gel. B,
immunoblot probed with anti-eIF4G
antibodies. C, immunoblot probed with anti-eIF4A. Lanes U and S represent eIF4F samples either untreated or treated with
protease 2A, respectively, but not subjected to
m
GTP-Sepharose chromatography. FT, flow-through
(unbound) fractions. m
GTP, fractions
eluted with m
GTP.
A similar analysis of eIF4F treated with L
protease was performed (Fig. 4). In this case, proteins in
column fractions were resolved by SDS-PAGE and visualized by either
silver staining (A) or immunoblotting with
anti-eIF4G (B) or
anti-eIF4G
antibodies (C). (The fact
that cp
stains much more strongly than cp
with
silver has been documented previously(15) , but the reason is
not known; it may be due to groups of charged amino acid residues
observed in the cp
portion of eIF4G (43) or
posttranslational modifications which introduce hydrophylic or charged
groups.) cp
, fragments of cp
, and eIF4A were
not retained on the column but were found in the flow-through fractions (A and B, lanes 1-3). cp
was also
detected in the flow-through (A, lanes 1-3). Most of the
cp
, however, was retained on the column and co-eluted with
eIF4E (A and C, lanes 6-8), indicating that the
eIF4E-binding site is contained within this region of eIF4G. The fact
that some of the cp
and cp
are found in the
flow-through as well (lanes 1-3) may indicate
dissociation from eIF4E during chromatography.
Figure 4:
mGTP-Sepharose column
fractionation of eIF4F treated with FMDV protease L. eIF4F was
incubated with protease and fractionated on m
GTP-Sepharose
as in Fig. 3except that L protease was used instead of 2A
protease (see ``Experimental Procedures''). A,
silver-stained gel. B, immunoblot of the same gel probed with
anti-eIF4G
antibodies. C, immunoblot
of the same gel probed with anti-eIF4G
antibodies.
In order to define
more precisely the region of eIF4G that binds to eIF4E, we determined
the L protease cleavage site at the cp/cp
junction. Fragments of eIF4G produced by a brief digestion with L
protease were subjected to reverse phase HPLC on a C4 column (Fig. 5). The peaks in fractions 40-50 contained cp
and cp
and were well resolved from the peak in
fractions 51-56, which contained cp
. Although
cp
and cp
eluted in the same general region,
it was possible to resolve the majority of the cp
(fractions 44-47) from the cp
(fractions
40-43). cp
was subjected to N-terminal sequence
analysis by automated Edman degradation (Table 1). Alignment of
the resultant sequence with the rabbit polypeptide sequence determined
by cDNA cloning indicated that L protease cleaves at
Lys
-Arg
, which corresponds to
Lys
-Arg
in human eIF4G. This indicates that
the eIF4E-binding site lies between amino acid residues 319 and 479.
Figure 5:
Purification of the cp
fragment by reverse phase HPLC. L protease-treated eIF4F was
fractionated by reverse phase HPLC on C4 as described under
``Experimental Procedures.'' Equal aliquots from the
indicated fractions were subjected to SDS-PAGE on 10% gels. A,
elution pattern of cp
, cp
, and
cp
. B, silver-stained gel of column fractions. C, immunoblot of the same gel probed with
anti-eIF4G
antibodies.
Knowledge of this site allows a comparison of the true molecular
masses of cp and cp
to the SDS-PAGE-derived
masses. The calculated molecular mass for cp
based on this
cleavage site is 32.5 kDa, but the observed mobility on SDS-PAGE for
cp
bands is 80-100 kDa. Also, cp
is
heterogeneous, whereas cp
is not (Fig. 5B). cp
migrates as a 28-kDa
polypeptide despite its true molecular mass of 18 kDa. This indicates
that cp
is responsible for most of the heterogeneity and
aberrant mobility of eIF4G.
Knowledge of the
cp/cp
junction also allows one to make
conclusions about further cleavage products of cp
. The
size difference between cp
and cp
is
2-4 kDa, but both are recognized by the
anti-eIF4G
antibodies ( Fig. 1and Fig. 5). This epitope is located only nine amino acid residues
(1.2 kDa) from the N terminus of cp
. The fact that
cp
is recognized by anti-eIF4G
antibodies suggests that it has the same N terminus as cp
and hence that the cleavage converting cp
to
cp
is at its C terminus (see Fig. 2).
The eIF3eIF4F complex was isolated, digested with either 2A or
L protease, and the products subjected to sedimentation analysis (Fig. 6). The sedimentation of eIF3 was indicated by
immunoreactivity of the 110-kDa eIF3
subunit. In the absence of
protease, eIF4G and eIF3
sedimented at approximately 18 S with
the eIF3
eIF4F complex (A, lanes 6-9). The majority
of the eIF4A (70%) co-sedimented with this complex as well, although
some apparently dissociated during sedimentation. Cleavage of eIF4G
disrupted the eIF3
eIF4F complex. When eIF4G was cleaved by 2A
protease, the two fragments of eIF4G did not co-sediment. Rather, all
of the cp
sedimented near the top of the gradient (B,
lanes 1-2), whereas all of the cp
and most (75%)
of the eIF4A co-sedimented with eIF3 (lanes 6-9). This
indicates that cp
and eIF4A remain associated with eIF3
after 2A cleavage of eIF4G, but cp
does not.
Figure 6:
Ultracentrifugal fractionation of the
protease-treated eIF3eIF4F complex. The eIF3
eIF4F complex
was incubated in the absence (A) or presence of the 2A (B) or L (C) proteases, layered onto 15-30%
sucrose gradients, and centrifuged as described under
``Experimental Procedures.'' Gradients were fractionated and
aliquots subjected to SDS-PAGE on 8.5% gels. 3
,
immunoblot probed with anti-eIF3 antibodies; 4A, immunoblot
probed with anti-eIF4A antibodies; 4G,
cp
, cp
, and cp
, immunoblot probed with
anti-eIF4G
antibodies; cp
, immunoblot probed with
anti-eIF4G
antibodies; cp
, immunoblot probed with
anti-eIF4G
antibodies. Lane S represents the starting material which was layered onto each
gradient. Fraction 1 corresponds to the top of the
gradient.
Limited
digestion of eIF4G by L protease disrupted its interaction with both
eIF4A and eIF3 (C). Whereas all remaining intact cp and some eIF4A still co-sedimented with eIF3 (lanes
5-8), most of the eIF4A (80%), as well as all of the
cp
and cp
, shifted to the top of the gradient (lanes 1-4). The proportion of eIF4A at the top of the
gradient was approximately equal to the proportion of cp
cleaved. Longer digestion with L protease resulted in complete
cleavage of cp
and all of the eIF4A shifting to the top of
the gradient (data not shown). The cp
fragment
co-sedimented with eIF3 (lanes 6 and 7), whereas
cp
and cp
were released and sedimented near
the top of the gradient (data not shown). These results suggest that
the eIF3-binding site is located within cp
.
Since eIF4A
co-sedimented with cp, the eIF4A binding site is likely to
be contained within this region. The fact that eIF4A shifts to the top
of the gradient when cp
is further digested with L protease (C) could mean that either eIF4A is free or is bound to a
fragment of eIF4G which has shifted to the top of the gradient, e.g. cp
or cp
. To determine whether
eIF4A has affinity for cp
or cp
, we
covalently attached purified anti-eIF4G
antibody to agarose beads and used this immunoaffinity resin to
test for physical association between eIF4A and fragments of eIF4G. L
protease was incubated with either eIF4A alone (Fig. 7, lane
1) or eIF4A mixed with eIF4F (lanes 2 and 3),
the reactions subjected to immunoadsorption, and the bound fractions
analyzed (lanes 4-6). (eIF4A was added to the eIF4F
preparation because eIF4F prepared by the method of Lamphear and
Panniers (25) is substoichiometric for eIF4A.) The immobilized
antibody bound cp
, cp
, and cp
as
expected (A, cf.lanes 2 and 3 with lanes 5 and 6); the strong band below cp
migrating at 50 kDa is the highly immunoreactive IgG heavy chain,
which probably results from insufficient removal of the immunoaffinity
resin from eluted samples). eIF4A alone was not bound to the
immunoaffinity resin (B, lane 4) unless eIF4G fragments were
present (B, lanes 5 and 6). In a similar experiment
in which the digestion with L protease was continued until all of the
cp
was converted to cp
, eIF4A was also
retained on the immunoaffinity resin (data not shown), indicating that
the
4 kDa which is removed from cp
by the secondary L
protease cleavage does not contain the eIF4A binding site.
Figure 7:
Immunoadsorption of L protease-treated
eIF4F with immobilized anti-eIF4G antibodies.
eIF4A (2 µg, lanes 1 and 4) or eIF4A plus eIF4F
(2 µg each, lanes 2, 3, 5, and 6) were incubated
with L protease in 50 µl of buffer D
. Aliquots (5
µl) were removed for gel analysis (lanes 1-3) and
the remainder subjected to immunoadsorption on an
anti-eIF4G
resin as described under
``Experimental Procedures.'' The eluate (bound fraction) was
then analyzed by SDS-PAGE on 8.5% gels (lanes 4-6). A, 12 µg of protease were used and incubation was for
either 30 min (lanes 1, 2, 4, and 5) or 90 min (lanes 3 and 6). The immunoblot was probed with
anti-eIF4G
antibodies. The band migrating at
50 kDa in lanes 5 and 6 is rabbit IgG heavy
chain, which contaminates some of the eluates. B, same as A except the immunoblot was probed with anti-eIF4A antibodies.
In this case the IgG heavy chain does not react, since the secondary
antibody used was goat anti-mouse. C, same as A except incubation with protease was for 30 min, 3 µg of
protease were used in lanes 1, 2, 4, and 5, and 6
µg were used in lanes 3 and 6. Also, the
immunoblot was probed with anti-eIF4G
antibodies.
As a
control, the anti-eIF4G antibody was used to
test whether cp
, cp
, or cp
were
bound to the immunoaffinity resin. If this were the case, e.g. that cp
remained bound to cp
after
proteolytic cleavage of cp
, it would not be possible to
distinguish whether eIF4A were bound to cp
or
cp
. The experiment indicated that cp
,
cp
, or cp
, though detectable in the starting
material (Fig. 7C, lanes 2 and 3), was not
retained by the resin (lane 5). (Contamination by the IgG
heavy chain did not permit analysis of cp
in lane
6, but cp
or cp
were clearly absent.)
cp
, on the other hand, which contains the epitope for both
anti-eIF4G
and anti-eIF4G
antibodies, was detected in the bound fractions (Fig. 7C, lanes 5 and 6). Thus, the
immobilized anti-eIF4G
antibody selectively
adsorbs cp
and cp
, but not cp
,
cp
, or cp
. In a similar experiment using
anti-eIF4G
antibodies, it was determined that
cp
did not bind to the immunoaffinity resin (data not
shown). In summary, these results indicate that there is a binding site
for eIF4A in the cp
fragment of eIF4G.
Previous studies have established that eIF4G can form
complexes with initiation factor polypeptides whose activities are
associated with each of the events necessary for recruitment of host
cell mRNA for translation. These events are cap recognition, a property
of eIF4E (reviewed in (52) ); ATP-dependent unwinding of mRNA
secondary structure, which is a property of eIF4A alone but one which
is greatly enhanced by the presence of eIF4B and
eIF4G(53, 54) ; and ribosome binding, which requires
eIF3(55, 56, 57) . Purification of a high
salt ribosomal wash by gel filtration (23) or
mGTP-Sepharose chromatography (25) leads to
co-elution of eIF4A, eIF4B, eIF4E, eIF4G, and eIF3 in a large
macromolecular complex. The present study indicates that treatment with
proteases which have high specificity for eIF4G separates these major
functional activities into two complexes, one containing the cap
recognition function and one containing the helicase and ribosome
binding functions (Fig. 3, 4, 6, and 7). In vivo, the
2A and L proteases appear to carry out only the cleavage event which
generates cp
and cp
(12) . Therefore,
the activities of these fragments are most relevant to viral
replication. However, further proteolysis by L protease in
vitro, again specific for eIF4G, results in the separation of the
ribosome binding function (eIF3) and the helicase function (eIF4A; Fig. 6). This suggests that these three functional activities of
initiation factors involved in mRNA recruitment (cap binding, RNA
helicase, and ribosome binding) are brought together principally
through an interaction with eIF4G rather than with each other.
cp is the region of eIF4G responsible for binding eIF4E,
representing amino acid residues 1-486 of rabbit eIF4G in the
case of 2A protease cleavage (1-479 in the case of L protease).
Finer mapping with L protease narrows this region to amino acids
319-479. The criterion in either case is retention on
m
GTP-Sepharose. Previously it was reported that
poliovirus-induced cleavage products of eIF4G, as detected by a
monoclonal antibody, were retained on cap-analog columns, but the
location of the epitope was unknown (27, 28, 29) . With the availability of
antibodies to defined regions of eIF4G, it is now possible to conclude
that the monoclonal antibody used earlier recognizes the same cleavage
products as antipeptide antibodies against amino acid residues
327-416(43) . The validity of m
GTP-Sepharose
retention as a basis for assigning the eIF4E-binding region to cp
is reinforced by the observation that cp
in the
absence of eIF4E is not retained on
m
GTP-Sepharose(28) .
cp,
representing amino acids 480-1402, is the region of eIF4G which binds
to eIF4A and eIF3, as demonstrated by co-sedimentation (Fig. 6)
and immunoadsorption (Fig. 7). cp
contains the
eIF3-binding site, based primarily on co-sedimentation, but also
supported by the observation that eIF3 alters cleavage within cp
(Fig. 1C). cp
contains the eIF4A
binding site, since both eIF4A and cp
are released from
eIF3 by L protease and co-sediment (Fig. 6C). Also,
eIF4A specifically binds to the anti-eIF4G
affinity resin, but only in the presence of cp
or
larger fragments containing cp
(Fig. 7B).
Putting these two sets of data together suggests a domain model for
eIF4G wherein the cap recognition function is in the N-terminal
one-third of the molecule and the unwinding and ribosome binding
functions are in the C-terminal two-thirds (Fig. 8). The region
separating the N- and C-terminal domains may be a flexible hinge or
loop which is more exposed to proteases. In support of this prediction,
analysis of secondary structure motifs suggests that this region has a
high probability of -turns(58) . Furthermore, a synthetic
peptide based on a different portion of the eIF4G sequence is cleaved
by HRV2 protease 2A(17) , but this site is not cleaved in
intact eIF4G(15) , suggesting that it is in a region of the
molecule which is not as accessible as the putative hinge region.
Finally, fragments of eIF4G similar in size to cp
are
observed upon raising Ca
levels in extracts of
uninfected cells, suggesting that the junction between cp
and cp
is more susceptible to normal intracellular
proteases (59) .
Figure 8:
A domain model for eIF4G in cap-dependent
and -independent initiation of translation. A model for the
involvement of eIF4G (4G) in cap-dependent and -independent
initiation is shown, based on the binding sites on eIF4G for eIF4E (E), eIF4A (A), and eIF3 (3). A,
cap-dependent initiation. Only one of the current models for
cap-dependent initiation, the stepwise assembly model, is depicted (see
text). eIF4E binds to the mRNA cap, whereas eIF4G binds to eIF3 on the
43 S initiation complex. The protein-protein interaction between eIF4E
and eIF4G brings the mRNA to the ribosome and the unwinding machinery,
represented by eIF4A. The wavy portion of mRNA indicates
secondary structure. The ability of eIF4G to bind eIF4A, eIF3, and
eIF4E is critical for mediating cap-dependent joining of mRNA to the
ribosome and positioning of the RNA helicase at the 5`-end of the mRNA. B, cap-independent initiation. During replication of rhino-,
entero-, and aphthoviruses, cleavage of eIF4G separates the
eIF4E-binding domain (cp) from the eIF3-
and eIF4A-binding domain (cp
). This
produces an altered 43 S initiation complex (*), disrupting the ability
of eIF4G to mediate cap-dependent recruitment of RNA to the ribosome
but not altering the binding of cp
to the ribosome or
helicase activity required for internal
initiation.
Is such a domain model compatible with
current ideas about cap-dependent initiation? Two models for the
mRNA-binding step of initiation have been proposed, here referred to as
the stepwise assembly model and the preformed complex model.
Recruitment of capped mRNAs requires the coupling of cap recognition,
unwinding, and scanning. The models differ primarily in which of these
functions occur on the ribosome. The division of eIF4G into functional
domains, however, is compatible with either model. The stepwise
assembly model states that eIF4E first recognizes the cap as a free
polypeptide, i.e. not in the eIF4F complex, whereas eIF4G is
present in the 43 S initiation complex prior to mRNA recruitment, bound
to eIF3 ((60) ; see Fig. 8). eIF4A, although largely
found in the postribosomal supernatant, may also be bound to eIF4G and
hence also be present in the 43 S initiation complex. The joining of
eIF4E to eIF4G brings mRNA to the 40 S ribosomal subunit and to the
unwinding machinery. In this model, the cp region would
serve to anchor eIF4G to the ribosome via eIF3 and provide a binding
site for eIF4A (and possibly eIF4B as well), whereas the cp
domain would provide a flexible arm to receive the incoming
eIF4E:mRNA complex (Fig. 8A). Cleavage of eIF4G by
viral proteases would permit eIF4E to bind the cap and the
eIF4E
mRNA complex to bind cp
(Fig. 8B,
upper portion), but these events would occur apart from the
unwinding machinery and the 40 S ribosomal subunit. Hence, no
recruitment of capped mRNA to the ribosome would result. The preformed
complex model states that eIF4F exists prior to formation of any
complexes between mRNA, initiation factors, or
ribosomes(3, 21, 22, 61) . This
eIF4F complex recognizes the cap of free mRNA and unwinds its secondary
structure. When a sufficiently long stretch of secondary structure is
unwound, the 40 S ribosomal subunit binds and begins scanning. A domain
structure for eIF4G as proposed here would suggest that eIF4E, bound to
the cp
region of eIF4G, first recognizes the cap and
directs the mRNA to the unwinding machinery which is bound to the
cp
region. Cleavage of eIF4G by viral proteases would yield
separate complexes capable of cap recognition and unwinding, but since
unwinding would not be directed to the 5`-end of the mRNA, ribosome
binding to single-stranded RNA would be frequently unproductive.
Is
such a domain model for eIF4G compatible with current ideas about
cap-independent initiation? Translation of picornaviral RNA requires
eIF4A, and inactive variants of eIF4A have a dominant negative effect
on translation of both capped and picornaviral mRNAs(36) . This
inhibition can be overcome by the addition of normal eIF4A, but the
addition of eIF4F is six times more effective, underscoring the
importance of eIF4G for unwinding. Binding of picornaviral RNA to the
ribosome requires an internal ribosome entry site sequence (reviewed in (62) ). As eIF3 is involved in mRNA binding to ribosomes (see
above), it must be presumed that translation of picornaviral RNA
requires eIF3. In summary, translation of picornaviral RNA requires
factors involved in unwinding (eIF4A, eIF4B) and ribosome binding
(eIF3) but not cap recognition (eIF4E). Cleavage of eIF4G by
picornaviral proteases separates the functions required for
cap-independent translation (those attached to cp) from
those which are not (those attached to cp
; see Fig. 8B). It is interesting in this light that the
primary cleavage sites in eIF4G for 2A protease (15) and L
protease (12) are only seven amino acid residues apart, despite
the fact that the proteases are structurally distinct and that the
amino acid sequences surrounding the cleavage sites bear little
similarity. Thus, there has been evolutionary conservation among the
entero-, rhino-, and aphthoviruses of function (separation of cp
from cp
) rather than recognition of a specific amino
acid sequence.
cp is thus likely to contain the
functions required for cap-independent initiation. A binding site for
eIF3 in cp
permits attachment to the ribosome. Consistent
with this, we have observed that cp
sediments with
ribosomes in a micrococcal nuclease-treated rabbit reticulocyte lysate
(data not shown). A binding site for eIF4A in cp
suggests
that unwinding functions can take place in the absence of
cp
. The unwinding activity eIF4A is greatly enhanced when
it is present in the eIF4F complex(53, 54) ; based on
the location of the eIF4A binding site, it is likely that this
stimulatory activity of eIF4G resides in cp
. Also, there is
a proposed RNA-binding region in eIF4G which is within
cp
(63) , and this region may be important for
recruitment of mRNAs to the ribosome or stabilization of mRNA-ribosome
interactions during translation of cellular and viral mRNAs. The
proposed requirement of cp
for internal initiation may
explain a paradoxical observation: picornavirus infection
``inactivates'' eIF4F(6, 64) , yet eIF4F has
been shown to stimulate internal cap-independent initiation from viral
sequences(34, 35, 65) . The proposed domain
model would state that the added eIF4F is supplying functions contained
in the cp
region which are needed for cap-independent
translation; eIF4F is ``inactivated'' by 2A or L protease
only with respect to cap-dependent translation. Based on this
consideration, one would predict that cp
alone would
stimulate cap-independent initiation through its eIF3, eIF4A, and
possibly RNA binding properties.
Note Added in Proof-Mader et al. (Mader, S., Lee, H., Pause, A., and Sonenberg, N.(1995) Mol. Cell. Biol., in press) have recently shown in agreement with our results that eIF4E interacts with the N-terminal portion of eIF4G. They have mapped the eIF4E-binding region to within amino acids 409-457 and have identified a conserved motif between amino acids 413-424 that is important for the interaction.