Molecular mechanisms of translation initiation in eukaryotes.

Abstract

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

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Colloquium
Molecular mechanisms of translation initiation
in eukaryotes
Tatyana V. Pestova*†, Victoria G. Kolupaeva*, Ivan B. Lomakin*, Evgeny V. Pilipenko*‡§, Ivan N. Shatsky†,
Vadim I. Agol†‡, and Christopher U. T. Hellen*¶
*Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, NY 11203;†A. N. Belozersky
Institute of Physico-chemical Biology, Moscow State University, Moscow 119899, Russia; and‡Institute of Poliomyelitis and Viral Encephalitides,
Russian Academy of Medical Sciences, Moscow Region 142782, Russia
Translation initiation is a complex process in which initiator tRNA,
40S, and 60S ribosomal subunits are assembled by eukaryotic
initiation factors (eIFs) into an 80S ribosome at the initiation codon
of mRNA. The cap-binding complex eIF4F and the factors eIF4A and
eIF4B are required for binding of 43S complexes (comprising a 40S
subunit, eIF2?GTP?Met-tRNAi and eIF3) to the 5? end of capped
mRNA but are not sufficient to promote ribosomal scanning to the
initiation codon. eIF1A enhances the ability of eIF1 to dissociate
aberrantly assembled complexes from mRNA, and these factors
synergistically mediate 48S complex assembly at the initiation
codon. Joining of 48S complexes to 60S subunits to form 80S
ribosomes requires eIF5B, which has an essential ribosome-depen-
dent GTPase activity and hydrolysis of eIF2-bound GTP induced by
eIF5. Initiation on a few mRNAs is cap-independent and occurs
instead by internal ribosomal entry. Encephalomyocarditis virus
(EMCV) and hepatitis C virus epitomize distinct mechanisms of
internal ribosomal entry site (IRES)-mediated initiation. The eIF4A
and eIF4G subunits of eIF4F bind immediately upstream of the
EMCV initiation codon and promote binding of 43S complexes.
EMCV initiation does not involve scanning and does not require
eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some
EMCV-likeIRESsrequiresadditionalnoncanonicalinitiationfactors,
which alter IRES conformation and promote binding of eIF4A?4G.
Initiation on the hepatitis C virus IRES is even simpler: 43S com-
plexes containing only eIF2 and eIF3 bind directly to the initiation
codon as a result of specific interaction of the IRES and the 40S
subunit.
T
60S ribosomal subunits into an 80S ribosome in which Met-
tRNAi is positioned in the ribosomal P site at the initiation
codon.Thecomplexinitiationprocessthatleadsto80Sribosome
formation consists of several linked stages that are mediated by
eukaryotic initiation factors. These stages are:
(i) Selection of initiator tRNA from the pool of elongator
tRNAs by eukaryotic initiation factor (eIF)2 and binding of an
eIF2?GTP?Met-tRNAiternary complex and other eIFs to the
40S subunit to form a 43S preinitiation complex.
(ii) Binding of the 43S complex to mRNA, which in most
instances occurs by a mechanism that involves initial recognition
of the m7G cap at the mRNA 5?-terminus by the eIF4E
(cap-binding) subunit of eIF4F. Ribosomes bind to a subset
of cellular and viral mRNAs as a result of cap- and end-
independent internal ribosomal entry.
(iii) Movement of the mRNA-bound ribosomal complex along
the 5? nontranslated region (5?NTR) from its initial binding site
to the initiation codon to form a 48S initiation complex in which
the initiation codon is base paired to the anticodon of initiator
tRNA.
ranslation of mRNA into protein begins after assembly of
initiatortRNA(Met-tRNAi),mRNA,andseparated40Sand
(iv) Displacement of factors from the 48S complex and joining
of the 60S subunit to form an 80S ribosome, leaving Met-tRNAi
in the ribosomal P site.
Research in our laboratory has addressed the molecular
mechanisms of these different stages in translation initiation and
the means by which they are bypassed during initiation by
internal ribosomal entry. We have reconstituted each of these
stages in vitro using purified translation components to identify
the minimum set of eIFs that is required for each stage and to
provide a framework for more detailed mechanistic analysis.
Factor Requirements for Ribosomal Attachment and Scanning of 43S
Ribosomal Complexes on ?-Globin mRNA. The initiation codon of a
eukaryotic mRNA is normally the first AUG triplet downstream
of the 5?-terminal cap and is usually separated from it by 50–100
nt. After cap-mediated attachment to mRNA, a 43S complex is
thought to scan downstream from the 5?-end until it encounters
the initiation codon. We used native capped ?-globin mRNA as
a model in in vitro reconstitution experiments to address three
basic questions. (i) Which eIFs are required for a 43S complex
to bind capped mRNA? (ii) Which eIFs are required for the
boundcomplextomovedownstreamtotheinitiationcodon?(iii)
How does the scanning 43S complex recognize and reject
mismatched interactions between the Met-tRNAi anticodon,
and triplets in the 5? NTR until the correct initiation codon is
reached and recognized? In these experiments, the position of
the leading edge of bound ribosomal complexes on mRNA was
mapped by primer extension inhibition (‘‘toeprinting’’). The
estimated length of the mRNA-binding cleft in 40S subunits is
?30nt, and 48S complexes usually yield toeprints at positions
?15 ? ?17 downstream of the A of the initiation codon.
Ribosomal binding at the 5?-end of the mRNA required eIF3,
the eIF2?GTP?Met-tRNAicomplex, ATP, and the eIF4F cap-
binding complex, and was enhanced by eIF4B (1). eIF4F is a
heterotrimeric factor, and its eIF4A (ATP-dependent RNA
helicase) and eIF4E subunits and the eIF4G550–1090fragment of
its 1,560-amino acid eIF4G subunit constitute the core of eIF4F
This paper was presented at the National Academy of Sciences colloquium, ‘‘Molecular
Kinesis in Cellular Function and Plasticity,’’ held December 7–9, 2000, at the Arnold and
Mabel Beckman Center in Irvine, CA.
Abbreviations: eIF, eukaryotic initiation factor; 5? NTR, 5? nontranslated region; IRES,
internal ribosomal entry site; EMCV, encephalomyocarditis virus; FMDV, foot-and-mouth
disease virus; TMEV, Theiler’s murine encephalitis virus; BVDV, bovine viral diarrhea virus;
CSFV, classical swine fever virus; HCV, hepatitis C virus; ITAF, IRES transactivating factors;
PTB,pyrimidinetract-bindingprotein;RRL,rabbitreticulocytelysate;GMP-PNP,guanosine
5?-[?,?-imido]triphosphate.
§Present address: Department of Neurology, University of Chicago Medical Center, Chi-
cago, IL 60637.
¶To whom reprint requests should be addressed at: Department of Microbiology and
Immunology, State University of New York Health Science Center at Brooklyn, 450
Clarkson Avenue, Box 44, Brooklyn, NY 11203. E-mail: chellen@netmail.hscbkyn.edu.
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sufficient for efficient ribosomal attachment to capped mRNA
(2). This fragment of eIF4G binds both eIF4E and eIF4A and
probably coordinates their activities so that a cap-proximal
region of mRNA is unwound and is thus rendered accessible to
an incoming 43S complex so it can bind productively. The
molecular interactions that enable the incoming 43S complex to
bind this ‘‘prepared’’ template are not known but are thought to
involveinteractionoftheeIF3componentof43Scomplexeswith
cap-associated eIF4G. The bound ribosomal ‘‘complex I’’ was
arrested in a cap-proximal position and did not reach the
initiation codon (Fig. 1A).
Two additional activities present in rabbit reticulocyte lysate
(RRL) enabled 43S complexes to reach the initiation codon,
forming ‘‘complex II’’ without being arrested at the initial
binding site (Fig. 1B). These small factors were purified and
identified by sequencing as eIF1 (13.5 kDa) and eIF1A (19 kDa)
and could be functionally replaced by corresponding recombi-
nant polypeptides. These two factors acted synergistically; eIF1A
without eIF1 enhanced eIF4F-mediated binding of 43S com-
plexes to mRNA but did not enable these complexes to reach the
initiation codon, whereas eIF1 without eIF1A reduced the
prominence of the cap-proximal complex I and promoted for-
mation of low levels of 48S complexes. The interaction with
mRNA of 48S complexes assembled in the absence of eIF1A
differed subtly from complexes formed in their presence, in that
only two (?16–?17) rather than three toeprints (?15–?17)
wereapparent.eIF1Athereforeincreasesthecompetenceof43S
complexes to bind mRNA and the processivity of scanning
43S?eIF1?mRNA complexes. eIF1A also stabilizes binding of
the ternary complex to 40S subunits in the absence of mRNA (3,
4), presumably by an allosteric mechanism, because it is not
known to interact directly with any component of the ternary
complex. This stabilization by eIF1A is weak but might be
indicative of a role for eIF1A in ensuring that initiator tRNA and
mRNA adopt the correct relative orientation on the scanning
ribosomal complex.
eIF1A comprises an oligonucleotide-binding (OB) ?-barrel
fold that closely resembles prokaryotic initiation factor IF1 (and
corresponds to the region of sequence homology between them)
and an additional C-terminal domain (4). The experimentally
determined RNA-binding surface of eIF1A is large, extending
over the OB fold and the adjacent groove leading to the second
domain. Mutations at multiple positions on this surface resulted
in a reduced ability of eIF1A to promote assembly of 48S
initiation complexes at the initiation codon. The RNA ligand for
eIF1A is not known, but by analogy with IF1 (5), eIF1A might
bind 18S ribosomal RNA in the ribosomal A site.
In the absence of eIF1 and eIF1A, the mRNA-binding cleft on
40S subunits appears to be open, because they can bind mRNA
in an end-independent manner during initiation by internal
ribosomal entry (see below). eIF1 and eIF1A may contribute to
the correct interactions of components of the 43S complex with
mRNA that enable it to enter a processive mode, for example by
closing this cleft directly or indirectly and possibly even by
forming part of the channel on the 40S subunit through which
mRNA moves during ribosomal scanning.
Experiments done by using competitor mRNAs indicated that
complex I cannot be ‘‘chased’’ directly into complex II and is
therefore not its immediate precursor. Complex I is aberrantly
assembled (because it is arrested at a non-AUG triplet and is
unable to scan to the initiation codon) and is intrinsically
unstable. eIF1 and eIF1A together (but not individually) pro-
mote dissociation of complex I and enable the released 43S
complex to rebind mRNA in a competent state to scan to the
initiation codon (Fig. 1C). eIF1 alone is able to recognize and
destabilize ribosomal complexes incorrectly assembled by inter-
nal ribosomal entry (see below). Identification of this activity of
mammalian eIF1 is consistent with characterization of its yeast
homologue Sui1 as a monitor of translation accuracy. Mutations
in Sui1 allow aberrant initiation in vivo at non-AUG codons by
mismatch base pairing with Met-tRNAi(e.g., ref. 6). Determi-
nation of the solution structure of eIF1 by NMR (7) has revealed
that these mutated residues form part of a surface that is almost
perfectly conserved among all eIF1 homologues and that is likely
directly involved in initiation codon selection by eIF1.
In summary, we have determined the set of factors required
for binding of a 43S complex to a model native capped mRNA
and for it to scan to the initiation codon. These experiments were
done by using ?-globin mRNA, and it is possible that ribosomal
scanning on longer or more highly structured 5?NTRs may
require additional as-yet-unidentified factors, for example to
enhance processivity or to promote unwinding of stable second-
ary structures. Almost all aspects of the mechanism of ribosomal
scanning remain uncharacterized (8). For example, scanning is
an ATP-dependent process, but it is not known whether ribo-
somal movement itself involves hydrolysis of ATP or whether
chemical energy is required only to unwind secondary structure
in the 5? NTR to permit ribosomal movement by one-
dimensional diffusion from its initial 5?-terminal attachment
site. The ability to reconstitute this process in vitro will enable
this and other outstanding questions to be addressed.
Factor Displacement from the 48S Complex and Joining to a 60S
Subunit to Form Active 80S Ribosomes.The 48S complex assembled
at the initiation codon of ?-globin mRNA is bound by factors
that must be displaced before the 40S subunit?mRNA?Met-
Fig. 1.
48S ribosomal complexes at the authentic initiation codon of a conventional
cappedmRNA.The5?terminalm7Gresidueisshownasafilledblackcircle,the
5? NTR as a black line, and the ORF downstream of the AUG initiation codon
asablackrectangle.(A)InthepresenceofeIFs2,3,4A,4B,and4F,anaberrant
ribosomal complex (‘‘complex I’’) assembles at a cap-proximal position but is
unable to scan downstream to the initiation codon. (B) In the presence of eIFs
1,1A,2,3,4A,4B,and4F,48Sribosomalcomplexesassembleexclusivelyatthe
authentic initiation codon. (C) Addition of eIF1 and eIF1A to complex I
promotes its complete conversion to correctly assembled 48S complexes after
dissociation of complex I and rebinding of 43S ribosomal complexes in a
scanning-competent form.
The mechanism of action of eIF1 and eIF1A in promoting assembly of
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tRNAicomplex can join with a 60S subunit. Substitution of GTP
by guanosine 5?-[?,?-imido]triphosphate (GMP-PNP) (a nonhy-
drolyzable analogue) arrests initiation at the stage of 48S
complex formation, indicating that displacement of factors and
subunit joining both require hydrolysis of GTP bound to eIF2 in
48S complexes. GTP hydrolysis by eIF2 is activated by eIF5, a
49-kDa polypeptide that interacts specifically with eIF2 and eIF3
(9, 10).
Recent data suggest that eIF5 is a component of multifactor
complex comprising eIF1, eIF3, eIF5, and the eIF2?GTP?Met-
tRNAiternary complex that can exist free of the ribosome and
probably binds to it as a whole rather than sequentially (10). eIF5
binds strongly to eIF2 but induces its GTPase activity only when
eIF2 is associated with the 40S subunit. GTP hydrolysis, which
leads to dissociation of eIF2-GDP, is thought to be induced in
response to base pairing between the initiation codon and the
anticodon of Met-tRNAi, thereby ensuring stringent selection of
the initiation codon during the scanning process (11).
Until recently, the hydrolysis of eIF2-bound GTP was con-
sidered the only requirement for the joining of a 60S subunit to
the 48S complex (see ref. 12 for a review). However, we found
that addition of 60S subunits and recombinant eIF5 to 48S
complexes assembled on globin mRNA did not lead to formation
of 80S ribosomes (13). A partially purified ribosomal salt wash
fraction from mouse ascites cells was active in promoting 80S
ribosome assembly and was therefore used as a source for
purification of additional factors. We purified two proteins to
apparent homogeneity, which together but not separately were
able to mediate assembly of 48S complexes and 60S subunits into
80S ribosomes. The smaller (49 kDa) protein could be function-
ally replaced by recombinant eIF5. The second protein had an
apparent molecular mass of 175 kDa, and its N-terminal se-
quence identified it as a mouse homologue of prokaryotic
initiation factor IF2 (Fig. 2). A role for a eukaryotic homologue
of IF2 was first revealed by studies in yeast (14). Analysis of
polyribosome profiles showed that deletion of the yeast IF2
homologue led to a reduction in formation of larger polysomes
and an accumulation of inactive 80S ribosomal particles, and in
vitro translation assays confirmed that this deletion led to a
defect in translation initiation on the majority, if not all, cellular
mRNAs. This defect could be rescued by adding back purified
recombinant protein (14). These results indicated that this
protein is a general translation factor in yeast. Human, Drosoph-
ila, and archaeal homologues have also been identified (15, 16).
In light of its function in subunit joining, we named this factor
eIF5B (13). Recombinant human eIF5B587–1220lacking amino
acids 1–586 could substitute for yeast eIF5B in vivo (15) and for
native mammalian eIF5B in subunit joining in our in vitro
reconstitution experiments (13). It is almost certain that eIF5B
is a protein that was previously implicated in subunit joining but
subsequently erroneously discounted as an inactive contaminant
of eIF5 (12).
Puromycin resembles the 3?-end of aminoacylated tRNA and
can bind to the ribosomal A site to react with Met-tRNAiin the
P site to form methionylpuromycin. This reaction mimics for-
mation of the first peptide bond, and we therefore used it to
confirm that 80S ribosomes assembled by using eIF5 and eIF5B
were active. Assembly of 48S complexes on AUG triplets is much
simpler than on native mRNA, because it involves neither
5?-end-dependent attachment nor scanning. 48S complex for-
mation on AUG triplets requires only a 40S subunit and the
eIF2?GTP?Met-tRNAicomplex,whichenabledustoinvestigate
the influence of other factors on the requirements for subunit
joining (13). A requirement for both eIF5 and eIF5B for 80S
assemblywasapparentonlywhen48Scomplexeswereassembled
by using eIF1, eIF1A, eIF2, and eIF3. These four factors are all
normally associated with a 48S complex at the initiation codon.
Individually, eIF5 and eIF5B were equally active in subunit
joining in reactions lacking eIF1 and eIF3, but inclusion of eIF1
and eIF3 together reduced the individual activities of both
eIF5 and eIF5B. The requirement for both eIF5 and eIF5B in
these circumstances indicates that they have complementary
functions.
Hydrolysis of GTP bound to 48S complexes is a prerequisite
for subunit joining and was therefore also compared in the
presence and absence of eIF1 and eIF3 (13). eIF5 and eIF5B
stimulated GTP hydrolysis by eIF2 equally when 48S complexes
contained only eIF1A and eIF2, but inclusion of eIF1 and eIF3
inhibited the stimulatory activity of eIF5B without affecting that
of eIF5. This effect can account for the reduced ability of eIF5B
to promote methionylpuromycin synthesis in the presence of
eIF1 and eIF3. We conclude that, although eIF5 is active in
inducing GTP hydrolysis on 48S complexes in the presence of a
full set of factors (including eIF1 and eIF3), this is insufficient
for subunit joining. Under these circumstances (when all factors
associated with 48S complexes are present, which corresponds to
the normal situation for initiation on capped mRNAs), eIF5B is
also required.
The central domain of eIF5B contains sequence motifs char-
acteristic of GTP-binding proteins (Fig. 2). By UV crosslinking,
we found that [32P]GTP bound directly to eIF5B independently
of ribosomal subunits, and that bound [32P]GTP exchanged
readily with unlabeled GTP, GMP-PNP, or GDP. eIF5B had no
detectable intrinsic GTPase activity, but its ability to hydrolyze
GTPwasactivatedby60Ssubunitsandconsiderablymoreby40S
and 60S subunits together. Interestingly, prokaryotic IF2 is also
Fig. 2.
from Homo sapiens (15), Drosophila melanogaster (16), and Saccharomyces
cerevisiae (14), archaeal IF2 from Methanococcus jannaschii and prokaryotic
IF2 from Escherichia coli (12). The percentages of amino acid identities to
human eIF5B in the N-terminal region of the protein, the GTP-binding do-
main,andtheC-terminalregionoftheproteinareshown.Theblackrectangle
in the schematic representation identifies the position of the GTP-binding
domains in these proteins with the indicated GTP-binding protein consensus
sequence motifs G1-G5 aligned with sequence motifs G1-G5 of E. coli IF2 and
human eIF5B. Numbers above the domains of eIF5B?IF2 proteins refer to the
amino acid residues in each protein; numbers below the aligned sequences
refer to the amino acid residues in G1–G5 motifs of human eIF5B.
Sequence and structural conservation of eukaryotic eIF5B proteins
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a GTPase that is specifically activated by large and small
ribosomal subunits together (17). This similarity between the
homologous factors eIF5B and IF2 suggests that ribosomal
activation of their GTPase activity may occur by a common
mechanism.
Binding of GTP to eIF5B may be required for it to adopt an
active conformation. To test this hypothesis, 48S complexes were
assembled with GTP, separated from unincorporated GTP by
gel filtration, and then incubated with eIF5, 60S subunits,
different nucleotides, and either full-length native eIF5B or
recombinant eIF5B587–1220. The degree of dependence of
eIF5B’sactivityin80SassemblyonbindingGTPwasdetermined
by the integrity of the protein. eIF5B587–1220 was completely
GTP-dependent, whereas native eIF5B retained low activity in
the absence of GTP but was nevertheless stimulated 3-fold by
GTP (T.V.P., unpublished work). This result suggests that eIF5B
adopts the active conformation required for subunit joining
when it binds GTP. eIF5B acts catalytically in the presence of
GTP, promoting multiple rounds of subunit joining. 80S com-
plexes were also formed by eIF5B bound to GMP-
PNP, but eIF5B-GMP-PNP acted stoichiometrically rather than
catalytically.
This defect in the activity of eIF5B in the presence of
GMP-PNP could be because hydrolysis of GTP bound to eIF5B
is required for the release of eIF5B from assembled 80S ribo-
somes, for the release of other factors, or for both. The propor-
tion of Met-tRNAiin 80S ribosomes assembled in the presence
of GTP (60%) that reacted with puromycin was significantly
greater than in complexes assembled by using GMP-PNP (8%).
Methionylpuromycin synthesis by purified 80S ribosomes assem-
bledinthepresenceofGTPwascompletelyinhibitedbyaddition
of eIF5B587–1220with GMP-PNP but not by either eIF5B587–1220
or GMP-PNP alone. This result indicates that eIF5B-GMP-PNP
can interact with preassembled 80S complexes, blocking their
ability to react with puromycin (13). The specific inhibition of
this reaction suggests that eIF5B binds to the ribosomal A site.
When ribosomal complexes assembled by using GTP were
resolved on sucrose density gradients, no eIF5B587–1220 was
detected on 40S, 48S, 60S, or 80S complexes. However, a large
amount of eIF5B587–1220was bound to 80S complexes assembled
in the presence of GMP-PNP. The inability of eIF5B587–1220to
hydrolyze GMP-PNP therefore locks the factor on 80S com-
plexes and renders them inactive in methionylpuromycin syn-
thesis. eIF1, eIF2, and eIF3 were detected in 48S complexes but
not in 80S complexes assembled with GTP or GMP-PNP. GTP
hydrolysis by eIF5B is therefore not required for the release of
these factors during subunit joining but is needed for release of
eIF5B itself. The inability of eIF5B587–1220?GMP-PNP to disso-
ciate from 80S ribosomes explains the requirement for stoichi-
ometric rather than catalytic amounts of this factor in assembly
reactions in the presence of GMP-PNP. Neither the stage during
the initiation process at which eIF1, eIF1A, eIF2, eIF3, and eIF5
are released nor the mechanism by which release occurs during
initiation on native mRNAs has yet been established.
Ribosomal subunit joining to form active 80S ribosomes that
are competent to begin elongation therefore involves two suc-
cessive GTP hydrolysis events: activation by eIF5 of hydrolysis of
eIF2-bound GTP and ribosome-activated hydrolysis of eIF5B-
bound GTP. Remarkably, eIF5B is a homologue of prokaryotic
IF2, which also mediates a similar subunit-joining step that also
involves ribosome-activated hydrolysis of factor-bound GTP.
Initiation of Picornavirus Translation by Internal Ribosomal Entry: The
Role of Canonical Initiation Factors. Picornavirus RNA genomes
are uncapped and have highly structured 5? NTRs that are
barriers to scanning ribosomes. Initiation on these mRNAs is
end-independent and is instead mediated by a ?400-nt internal
ribosomal entry site (IRES) in the 5? NTR (18, 19). The activity
of an IRES depends on its structural integrity, and even point
mutations can cause general or cell type-specific loss of function.
Picornavirus IRESs are divided into two major groups on the
basis of sequence and structural similarities (20, 21). One group
contains poliovirus and rhinovirus, and the other group contains
encephalomyocarditis virus (EMCV), Theiler’s murine enceph-
alomyelitis virus (TMEV), and foot-and-mouth disease virus
(FMDV). The EMCV and TMEV initiation codons are located
atthe3?borderoftheIRES,andribosomesbinddirectlytothem
without scanning (22, 23). In poliovirus, the initiation codon is
?160 nt from the 3? border of the IRES, and it is possible that
the ribosome reaches it either by scanning or by discontinuous
transfer (‘‘shunting’’) after initial attachment to the IRES (24).
Picornavirus infection often leads to shutoff of cap-mediated
translation initiation, for example by rhinovirus protease cleav-
age of eIF4G at R641?G642, such that the N-terminal domain
of eIF4G that binds eIF4E and the poly(A)-binding protein is
separated from the C-terminal domain that binds eIF3 and
eIF4A (25). This cleavage impairs eIF4F’s function in initiation
on capped mRNAs. However, as described below, this cleavage
yields a fragment of eIF4F that retains functions necessary for
picornavirus IRES-mediated initiation.
We reconstituted initiation in vitro on the EMCV IRES and
found that it is ATP-dependent and requires only eIFs 2, 3, and
either eIF4F or eIF4A and the central third of eIF4G to which
eIF4A binds (26, 27). The requirement for eIF4A and the
cognate domain of eIF4G is consistent with the profound
inhibition of EMCV translation caused by dominant negative
eIF4A mutant polypeptides (28). The inhibition caused by these
mutants is thought to be because of their failure to exit the eIF4F
complex and recycle efficiently, thereby trapping it in an inactive
form. In this model, eIF4A therefore plays its role in initiation
as part of a complex with eIF4G rather than as a singular
polypeptide.
48S complex formation was enhanced 4-fold by eIF4B and less
than 2-fold by the pyrimidine tract-binding protein (PTB), a
noncanonical mRNA-specific initiation factor (see below). To-
gether, these factors promoted 48S complex formation equally at
AUG834(the authentic initiation codon) and at AUG826(which
is virtually unused in vivo). Remarkably, inclusion of eIF1 in
assembly reactions or even addition of eIF1 to preformed
complexes led to dissociation of the ribosomal complex at
AUG826(13). This observation is consistent with the previously
noted function of eIF1 in enhancing the fidelity of initiation
codon selection. The principal difference between the factor
requirements for initiation on ?-globin mRNA and on the
EMCV IRES is that the latter has no requirement for eIF4E or
the fragment of eIF4G to which it binds and is therefore not
impaired by cleavage of eIF4G by viral proteases. eIF4E is a
major focus of mechanisms that regulate initiation of translation
in vivo (29). The EMCV IRES and other IRESs that do not
require eIF4E are therefore active in circumstances that lead to
inhibition of cap-mediated initiation by impairment of eIF4E
function.
A significant insight to the mechanism of initiation on the
EMCV IRES came from the observations that eIF4F bound to
the J-K domain of the EMCV IRES a little upstream of the
initiation codon (Fig. 3), and that this interaction is essential for
initiation (26). The essential central eIF4G722–949domain binds
specifically to the IRES, and its binding is strongly enhanced by
eIF4A (33). The interaction of eIF4G with this IRES may play
a role analogous to that of eIF4E on capped mRNAs, that is, to
recruit the eIF4F complex and associated factors and to promote
ribosomal attachment at a defined location on an mRNA.
The Role of IRES Transacting Factors in Initiation of Picornavirus
Translation. The activity of a number of picornavirus IRESs is
subject to cell-type-specific restriction: for example, the atten-
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uation of poliovirus vaccine strains is in part because of a defect
in translation in neuronal cells. Poliovirus and rhinovirus IRESs
mediate initiation of translation efficiently in HeLa and Krebs 2
cells, and their restricted activity in RRL in vitro can be
alleviated by deletion of the IRES or by supplementation of
RRL by ribosomal salt wash fractions from HeLa or Krebs cells
(e.g., refs. 34, 35). Translation mediated by poliovirus and
rhinovirus IRESs thus depends on the interaction with these
IRESs of noncanonical IRES transacting factors (ITAFs) that
are either absent from RRL or significantly less abundant in
RRL than in permissive cells. In early experiments to identify
ITAFs required for picornavirus IRES function, we and others
identified a 57-kDa protein that bound specifically to all picor-
navirus IRESs as the PTB, a cellular polypeptide that contains
four RNA-recognition motif -like domains (36–39).
The PTB dependence of initiation on the wild-type EMCV
IRES is small (27, 40) but was significantly enhanced by a single
nucleotide insertion in the eIF4G-binding site or by alteration of
the sequence downstream of the initiation codon (40). Foot-
printing analysis indicated that PTB binds multiple noncontig-
uous sites on the EMCV IRES (ref. 30; Fig. 3). Taken together,
these observations suggested a model in which binding of ITAFs
such as PTB stabilizes an IRES in an optimal conformation for
binding of essential factors and the 43S complex. Our analysis of
initiation on the related TMEV and FMDV IRESs provided
strong support for this hypothesis (31).
TMEV(GDVIIstrain)andFMDV(type01K)areneurotropic
and epitheliotropic picornaviruses, respectively. Their IRESs
are ?40% identical and are closely related to the EMCV IRES.
Substitution of the IRES in an infectious genomic TMEV clone
by that of FMDV strongly attenuated the neurovirulence of the
resulting chimeric virus without significantly affecting its ability
to replicate in cultured BHK-21 cells or to be translated in vitro
in RRL (31). We used biochemical reconstitution of the initia-
tion process on these mRNAs to define the molecular basis for
this cell type-specific difference in the function of these IRESs.
Initiation on the FMDV and TMEV IRESs had identical
requirements for canonical initiation factors to those described
above for EMCV. However, whereas initiation on the EMCV
IRES was only weakly stimulated by PTB, initiation on the
TMEV IRES depended strongly on PTB, and initiation on the
FMDV IRES required both PTB and a 45kDa ITAF (ITAF45).
We purified and sequenced ITAF45and found that it is identical
to a previously identified proliferation-dependent protein that is
not expressed in nonproliferating cells such as neurons (31). The
absence of this factor may account for the inability of the
chimeric TMEV virus to replicate in neurons.
The activities of PTB and ITAF45in promoting 48S complex
formation on the FMDV IRES were strongly synergistic. These
ITAFs bound to nonoverlapping sites on the IRES (Fig. 3) and
together caused localized conformational changes in it, specif-
icallyinregionsadjacenttothebindingsitefortheeIF4G?eIF4A
complex. The interaction of the IRES with the eIF4G?eIF4A
complex is essential for initiation and, significantly, this inter-
action was specifically enhanced by these two ITAFs. EMCV,
FMDV, and TMEV IRESs all bind to PTB and ITAF45, so it is
the requirement for them rather than their ability to interact that
differs as a consequence of sequence differences between these
IRESs. Similar observations have also been made for the second
group of picornavirus IRESs, members of which are also closely
related to each other yet also appear to have different ITAF
requirements. For example, poliovirus and rhinovirus IRESs
both bind to PTB, to the poly(rC)-binding protein 2 (PCBP2),
and to unr (35, 41). PTB contains four RNA-recognition motif-
likedomains,PCBP2hasthreeKHdomainsthatlikelyconstitute
its RNA-binding surface, and unr contains five cold-shock
RNA-binding domains. These polypeptides therefore all have
the potential to make multipoint interactions with these IRESs
andtostabilizetheirfoldinginanactiveconformation.However,
whereas initiation on the rhinovirus IRES depends on unr,
strongly enhanced by PTB and less responsive to PCBP2, the
poliovirus IRES depends on PTB and PCBP2 and does not
respond to unr (35).
Our analysis of initiation on EMCV-like IRESs suggests a
model that will likely be applicable to poliovirus-like IRESs and
possibly to some other viral and cellular IRESs. Specific binding
of eIF4F (or its eIF4G and eIF4A subunits) to the IRES is
required to mediate internal ribosomal entry and itself depends
on the eIF4F and ribosomal binding sites having the correct
conformation. The role of ITAFs is to bind an IRES to enable
it to attain or maintain this conformation, for binding both of
eIF4G?eIF4A and possibly of other components of the 43S
complex. The diversity of IRES sequences and structures leads
to the requirement for a variety of ITAFs.
Internal Initiation by Factor-Independent Binding of Ribosomes to the
Initiation Codon. The 5?NTRs of HCV and of the related classical
swine fever virus (CSFV) and bovine viral diarrhea virus
(BVDV) also promote translation by cap-independent internal
ribosomal entry (e.g., ref. 42). IRESs are defined solely by
functional criteria and cannot yet be predicted by the presence
of characteristic RNA sequence or structural motifs. As a rule,
there are no significant similarities between individual IRESs
(unless they are from related viruses). The related BVDV,
CSFV, and HCV IRESs are the best characterized members of
an IRES group that is wholly distinct from both the EMCV-like
and poliovirus-like groups of IRESs with regard to length,
sequence, and structure. We investigated initiation on BVDV,
CSFV, and HCV IRESs to determine whether all IRESs me-
diate internal initiation of translation by a single common
mechanism irrespective of sequence variation and, if not, to
characterize unique aspects of initiation on this group of HCV-
like IRESs.
The BVDV, CSFV, and HCV 5? NTRs are 385, 372, and 342
nt long, respectively, and although they differ from each other at
35–50% of base positions, many of these nucleotide differences
are covariant substitutions, indicative of conserved higher order
structure. Even minor mutations in structural elements substan-
tially reduced IRES activity, but this could in most instances be
restored by compensatory second site mutations that restored
Fig. 3.
H–L, showing binding sites for PTB (thick gray line) and ITAF45(black icosahe-
dron), as determined by footprinting (30, 31), and for the eIF4G?eIF4A com-
plex, as determined by footprinting (31, 32) and toeprinting (26, 27, 33). The
interaction of eIF4G?eIF4A with the J–K domain, which is essential for recruit-
mentofthe43Scomplextotheinitiationcodon,isenhancedbyPTBandITAF45
in an IRES-specific manner, as discussed in the text.
Schematic representation of EMCV, FMDV, and TMEV IRES domains
Pestova et al. PNAS ?
June 19, 2001 ?
vol. 98 ?
no. 13 ?
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