(Received for publication, April 14, 1995; and in revised form, June 12, 1995)
From the
Moloney murine leukemia virus (Mo-MuLV) genomic mRNA codes for two gag precursors by alternative initiations of translation. An AUG codon governs the synthesis of the retroviral capsid proteins precursor, whereas a CUG codon directs the synthesis of a glycosylated cell surface antigen, the gross cell surface antigen. Control of the relative synthesis of the two precursors is crucial for MuLV infectivity and pathology. Furthermore, the MuLV mRNA leader sequence is very long and should inhibit translation according to the classical scanning model. This suggests a different translation initiation mechanism allowing gag efficient expression.
We demonstrate, by using bicistronic vectors expressed in COS-7 cells, that the Mo-MuLV mRNA leader drives translation initiation by internal ribosome entry. We have localized the internal ribosome entry site (IRES) between the two initiation codons. This 126 nucleotide long IRES implies an oligopyrimidine tract located 45 nucleotides upstream of AUG codon. UV cross-linking and affinity chromatography experiments show that the PTB/p57 splicing factor specifically interacts with this oligopyrimidine tract.
The MuLV IRES controls alternative translation initiation by activating the capsid protein precursor expression. This gag translational enhancer could exist in other retroviruses.
The genomic RNA of retroviruses has the functions of genome, premessenger, and messenger. The discrimination between these three different functions is controlled by regulatory elements located in the RNA leader sequence. These elements, essential for reverse transcription, splicing, RNA dimerization, encapsidation, and translation, are compacted within an RNA sequence of less than 800 nucleotides(1, 2) . The different functions of the retroviral RNA have provided interesting models for the study of splicing and translation regulation of eukaryotic messengers in general.
The murine leukemia virus (MuLV), ()able to
infect a large range of mammalian cells when amphotropic, is used for
the construction of retroviral vectors in numerous strategies for gene
therapy. Its genomic mRNA possesses an original process of alternative
initiation of translation leading to the synthesis of two gag precursors(3, 4) : the AUG-initiated
Pr65
, precursor of the virion capsid proteins,
and the CUG-initiated Pr75
, precursor of a
glycosylated antigen, the gross cell surface antigen. The gross cell
surface antigen, never found in virions, is, however, essential for
MuLV spreading and pathogenicity (5, 6) . Thus, MuLV gag translation has to be subtly regulated to maintain a
correct level of both precursors. This implies that the relative use of
the two initiation codons is well controlled.
The existence of very long leader sequences in Mo-MuLV (7) and retrovirus in general suggests the existence of a translational control. Indeed, according to the classical cap-dependent scanning mechanism, translation initiation should be very inefficient as unwinding initiation factors would hardly be able to unwind the strong secondary structure of the leader(8, 9, 10) . This suggests that retroviruses, having conserved the long leaders required for replication and packaging, must have found a way to override inefficient ribosome scanning. One way would be the use of efficient transcription signals to flood the cells with mRNA. Another would be to render translation efficient by use of a translation initiation mechanism overcoming the scanning process. Consistent with this, are the pyrimidine-rich sequences found upstream from the AUG initiation codon of Mo-MuLV, which exhibit homologies with the polypyrimidine tracts reported for internal ribosome entry in picornavirus RNAs (11, 12, 13) .
The process of internal ribosome entry is an alternative to the classical ribosome entry process involving scanning from the 5`-capped end of the mRNA(10) . The first and most-studied example of such an original process is that of picornaviruses(14, 15) . The picornavirus RNA has no cap and possesses a long (600-1200 nucleotides) 5`-untranslated with multiple cryptic AUGs. Internal ribosome entry sites (IRES) have been found in several picornaviruses including poliovirus(16) , encephalomyocarditis virus (EMCV, 17, 18), foot-and-mouth disease virus(19) , human rhinovirus(20) , and hepatitis A virus(21) . The process has been further demonstrated for hepatitis C virus (22) and for cowpea mosaic virus, a plant comovirus(23) .
The finding of cellular factors bound to
IRESes and probably involved in the internal entry process suggested
that cellular messengers might also be
concerned(24, 25) . This hypothesis was confirmed by
the finding of IRESes in yeast TFIID and HAP4 transcription factors
mRNAs(26) , in drosophila antennapedia mRNA(27) , and
in human Bip and fibroblast growth factor 2 (FGF-2)
mRNAs(28, 29) . The case of FGF-2 is of particular
interest as its messenger possesses four initiation codons, and the
IRES promotes the synthesis of CUG-initiated forms that seem specific
to a range of transformed cells. ()
Numerous studies have produced insights into the mechanism of internal initiation of picornaviruses. The EMCV IRES, in particular, is a complex internal cis-acting element about 450 nucleotides in length that directs the ribosome to first contact the RNA at an AUG codon located at the 3`-end of the IRES, some 25 nucleotides downstream of an oligopyrimidine tract which is the only extended primary sequence motif common to all picornavirus IRESes(11, 12, 13) . Two trans-acting factors, the PTB/p57 splicing factor and the La autoantigen, have been identified as involved in the internal entry process of EMCV and poliovirus, respectively(24, 25, 30) .
The presence of
oligopyrimidine stretches upstream from the AUG-gag translation initiation codon of Mo-MuLV, and observation that
translation was cap-independent in vitro, ()incited
us to look for a mechanism of internal ribosome entry in Mo-MuLV mRNA.
We show here that the Mo-MuLV mRNA contains a 126 nt long IRES, located
between the two initiation codons. Translation from the AUG start codon
would thus occur by internal ribosome entry whereas translation from
the CUG start codon occurs by the cap-dependent scanning mechanism. The
IRES implies an oligopyrimidine tract located 45 nucleotides upstream
from the AUG codon. We also demonstrate the existence of specific
interactions of PTB/p57 splicing factor with the oligopyrimidine tract
of Mo-MuLV IRES.
Figure 1:
Expression of
monocistronic and bicistronic MuLV-CAT chimeric mRNAs in COS-7 cells. A, schematic representation of the chimeric constructs. Vector pMC, MuLV-CAT fusion under the control of CMV and T7
promoters. The MuLV-CAT fusion possessed the 671 5`-nucleotides of
Mo-MuLV sequence fused to CAT coding sequence, with the two Mo-MuLV
initiation codons in frame with CAT ORF (see ``Material and
Methods''). pBi CMC, bicistronic vector containing CAT
ORF upstream of MuLV CAT fusion. pHPBi CMC, derived from pBi
CMC, with addition of a 5`-hairpin (G = -40
kCal/mol). pMC HP1 and pMC HP284, derived from pMC,
with addition of a very stable hairpin (
G =
-80 kCal/mol) at the 5`-end or at 284 nt from the 5`-end,
respectively. B, COS-7 monkey cells were transiently
transfected with the vectors described in A. Cell extracts
were analyzed by Western immunoblotting (3 µg of total
proteins/lane) using anti-CAT antibodies, allowing the simultaneous
detection of both products from ORF1 and from ORF2, (see
``Materials and Methods''). To compare the translation level
at the Mo-MuLV codons obtained with mono- and bicistronic constructs,
the cell extract obtained from transfection with the monocistronic
plasmid pMC was diluted 2-, 4-, 8-, or 16-fold (as indicated in lanes 2-6). The results shown correspond to a
representative experiment which is repeated at least five times. The
plasmid used for each transfection assay is indicated on the top of
each lane. Mock (lane 1) corresponds to
non-transfected COS cells. Migration of CAT (ORF1) and of the
two MuLV-CAT fusion proteins are indicated by AUG CAT, AUG
MuLV-CAT, and CUG MuLV-CAT,
respectively.
Figure 3: Mapping of the IRES by progressive deletions in Mo-MuLV leader sequence. A, schematic representation of the different deletions of Mo-MuLV 5` that were carried out in the vector pBi CMC and pHPBi CMC (without or with hairpin, respectively). Only the pBi CMC series is schematized here. The name of each deleted vector corresponds to the number of nt that were deleted from Mo-MuLV 5`. B, COS-7 cells transfections and Western immunoblotting were performed as in Fig. 1, using the vectors described in A. The name of the vector used for each assay is indicated on the top of the lane. Absence or presence or hairpin is indicated by - (pBi series) or + (pHPBi series). Migration of ORF1 (AUG CAT) and ORF2 (AUG MuLV-CAT) products are indicated.
Figure 4: Identification of the IRES by site-directed mutagenesis in Mo-MuLV oligopyrimidine motifs. A, schematic representation of the bicistronic vector pBi CMC already used in Fig. 1and Fig. 2, and of the region 573 to 623 of Mo-MuLV RNA. The AUG codon and the nucleotides that were mutated are indicated in bold characters. The nucleotide substitutions introduced in the resulting bicistronic vectors pBi CMC m1 and pBi CMC m2 are indicated below. A version of these vectors with a 5`-hairpin derived from pHPBi CMC (see Fig. 1) was also constructed. B, schematic representation of the RNA secondary structure of the region 565-630 of Mo-MuLV RNA according to a previous report(35) . The AUG triplet and the mutated nucleotides are shown in bold characters. C, COS-7 cells transfections and Western immunoblotting were performed as in Fig. 1, using the vectors pBi CMC, pBi CMC m1, and pBi CMC m2 as well as their hairpin-containing version (pHPBi, see ``Materials and Methods''). The name of the vector used for each assay is indicated on the top of the lane. Absence or presence or hairpin is indicated by - (pBi series) or + (pHPBi series). Migration of ORF1 (AUG CAT) and ORF2 (AUG MuLV-CAT) products are indicated.
Figure 2:
Northern blotting of monocistronic and
bicistronic RNAs. COS-7 cells were transfected with mono- and
bicistronic vectors described in Fig. 1A. Total RNAs
were purified and analyzed by Northern blotting as described under
``Materials and Methods,'' using a P-labeled DNA
probe corresponding to CAT coding sequence. The plasmid used for each
transfection assay is indicated on the top of each lane. Lane 1 corresponds to mock transfected COS cells. Migration of 18 S and
28 S ribosomic RNAs is indicated as a size control. Migration of
monocistronic (Mono) and bicistronic (Bi) mRNAs are
indicated by arrows. The picture corresponds to 6 h exposure
of the filter at -80 °C.
pKS-PTB, the
plasmid used for in vitro transcription of PTB mRNA, was
constructed by subcloning a PCR fragment containing PTB cDNA, amplified
from the plasmid PE15 (gift of P. A. Sharp) into the HindIII
and BglII sites of vector Bluescript-pKS using
oligonucleotides 5`-CGGATCCAAGCTTACATGTTGGCCATGGACGGCATCGTCC-3` and
5`-GGGAGATCTCCTAGATGGTGGACTTGGAG-3` (5` and 3` of PTB ORF with HindIII and BglII sites, respectively). The plasmids
used for in vitro transcription of U1A mRNA and of U1 and
U1 RNAs were a gift from I. W. Mattaj.
DNAs were checked by sequencing with the dideoxy method.
Figure 5:
UV cross-linking of COS-7 cellular factors
to Mo-MuLV and EMCV IRESes. S10 extracts from COS-7 cells were
incubated with 10 counts/min of RNA probe corresponding to
MuLV IRES (fragment 495-671) (A) or EMCV IRES (nt
261-837) (B). Competition experiments were carried out
by addition of unlabeled RNA at a molar excess of 20-50-fold. UV
irradiation was performed as described under ``Materials and
Methods'' with an energy of 400,000 µJ/cm
at 254
nm, except for lanes 2 (A and B)
corresponding to an irradiation at 200,000 µJ/cm
.
Samples were treated with RNase ONE before analysis by SDS-PAGE. A, the probe was Mo-MuLV fragment 495-671, except for lanes 11, 12, and 14 for which the probe was
EMCV fragment 261-837 or mutant MuLV m2, as indicated on the top
of the lanes. Addition of competitors is indicated on the top of lanes 5-10. Lane 1 corresponds to RNA alone. Lanes 2 and 3 correspond to irradiations with
energies of 200,000 and 400,000 µJ/cm
, respectively. Lane 4 corresponds to sample treatment with proteinase K.
Migration of the size standards is shown. The sizes of the cross-linked
products are indicated by arrows. B, the probe was
EMCV fragment 261-837. Lanes 1-10 correspond to
the same treatment as in Fig. 5A. Lanes 11 and 12 correspond to a competition using the m2 mutant MuLV
fragment. Migration of the size standards is shown. Position of the PTB
is indicated by an arrow.
Figure 6:
Streptavidine acrylamide precipitation of
PTB/p57 protein with biotinylated RNA. S-Labeled U1A or
PTB protein was incubated for 1 h at 25 °C with different
biotinylated RNAs, as indicated on the top. The biotinylated
RNA
[
S]protein complexes were then
precipitated with streptavidine acrylamide beads and analyzed by
SDS-PAGE, as described under ``Materials and Methods.'' The
RNAs used were U1 (lanes 3 and 10), U1
B (lane 4), EMCV fragment 261-837 (lane 7), wild
type MuLV fragment 495-621 (lane 8), m2 mutant MuLV
495-621 (lane 9). Lanes 1 and 5 correspond to 20% of the input/assay; lanes 2 and 6 correspond to incubation of the proteins with the beads without
RNA.
In vitro translation in wheat germ
extract (Promega) was performed as described previously(31) ,
in the presence of [S]methionine (Amersham).
Northern blotting was performed as described
previously. DNA probes were labeled with [P]dATP
using a random priming kit (Promega). Total cellular RNA (1
µg/lane) was subjected to electrophoresis through 1.2%
formaldehyde-agarose gels, electrotransferred to nylon membrane, and
hybridized in the conditions described previously.
Western immunoblotting was performed as described previously(31) . The lysates were heated for 2 min at 95 °C in sodium dodecyl sulfate and dithiothreitol-containing sample buffer, separated in a 12.5% polyacrylamide gel, and transferred to a nitrocellulose membrane. CAT were immunodetected using rabbit polyclonal anti-CAT antibodies prepared in the laboratory (1/50000 dilution). Antibodies were detected using an enhanced chemiluminescence kit (Amersham).
CAT assays were performed as previously by using the diffusion-based CAT assay kit provided by NEN(31) .
For UV cross-linking
experiments, 1.10 counts/min of
P-labeled RNA
was incubated with 10 µg of S10 extract in buffer containing 5
mM HEPES, pH 7.6, 25 mM KCl, 2 mM
MgCl
, 3.8% glycerol, 0.02 mM dithiothreitol, and
1.5 mM ATP in a final volume of 10 µl at 30 °C for 15
min(25) . For competition experiments, cold competitor RNAs or
calf liver tRNA (Boehringer-Mannheim) were preincubated with the S10
extract for 15 min at 30 °C; then, 10
counts/min of
P-labeled RNA was added and the mixture was further
incubated at 30 °C for 10 min to allow complex formation. Samples
were then transferred to ice and irradiated using a UV Stratalinker
(Stratagene). They were fixed at a distance of 10 cm from the bulbs and
routinely irradiated with 2-400000 µJ/cm
at 254
nm. The samples were then treated with RNase ONE (10 units, Promega) at
37 °C for 30 min and, when indicated, with proteinase K (Sigma) at
37 °C for 20 min at a final concentration of 1 mg/ml.
Electrophoresis sample buffer was added and the samples were heated 2
min at 95 °C and loaded on a 10% SDS-PAGE (33) . The gel
was fixed in 30% methanol, 10% acetic acid for 30 min, dried on Whatman
paper, and autoradiographed.
The first strategy, termed the hairpin insertion strategy, was based on addition of a hairpin at the 5`-end or within the leader sequence of the messenger to block either ribosome binding or scanning, respectively(8, 9) . The second or bicistronic vectors strategy was based on addition of an open reading frame upstream of the messenger leader sequence and was expected to prevent expression of the open reading frame of interest unless preceded by an IRES(16, 17) .
We initially
constructed a fusion of the 5` first 671 nucleotides of Mo-MuLV
sequence (leader sequence plus the beginning of gag gene), with the CAT
coding sequence, under the control of a cytomegalovirus promoter (Fig. 1A, pMC). This construct was expected to
express both CUG and AUG-initiated MuLV-CAT fusion proteins in
transfected eukaryotic cells. In order to block the cap-dependent
ribosome entry by the hairpin insertion strategy, a stable hairpin
(G = -80 kCal/mol) was inserted either at
the 5` end or at position 284 from the 5` end of the MuLV-CAT fusion (Fig. 1A, pMC HP1 and HP 284).
Secondly, we constructed bicistronic vectors with the CAT coding
sequence upstream of the MuLV-CAT fusion (Fig. 1A, pBi CMC and pHPBi CMC). A 5`-hairpin (
G = -40 kCal/mol) was added to the vector pHPBi CMC
expected to inhibit specifically the cap-dependent
translation(28) . The advantage of bicistronic vectors with
tandem CAT reporter genes was the possibility of detecting expression
of both cistrons on the same blot, using anti-CAT antibodies.
Monkey COS-7 cells were transfected by the vectors described in Fig. 1A, and the expression of CAT and MuLV-CAT proteins was analyzed by Western immunoblotting (Fig. 1B). As expected, the monocistronic vector pMC gave two bands corresponding to the CUG and AUG-initiated MuLV-CAT proteins, respectively (lanes 2-6). In contrast, the two monocistronic vectors with hairpin insertions gave one band corresponding to the AUG-initiated protein (lanes 9 and 10). This feature was also observed for the bicistronic vectors (lanes 7 and 8): two bands were visible, corresponding to CAT (first cistron) and to AUG-initiated MuLV-CAT (second cistron), respectively. Furthermore, CAT but not MuLV-CAT expression decreased in the presence of the 5`-hairpin (lane 8), indicating that translation initiation at the AUG codon occurred independently of the RNA 5`-end.
One can argue that the second cistron of the bicistronic vector could be expressed from some unexpected form of monocistronic mRNA resulting from a cleavage of the bicistronic mRNA or from the use of a cryptic promoter between the two cistrons. To address this possible problem, RNA from COS-7 cells transfected by the different vectors was analyzed by Northern blotting (Fig. 2). Clearly the monocistronic and bicistronic mRNAs both migrated at the expected size. The pHPBi CMC RNA had a tendency to aggregate (probably because of the simultaneous presence of the MoMuLV RNA dimerization sequence and of the 5`-hairpin, 2). However the bicistronic constructs pBi CMC and pHPBi CMC did not give rise to any monocistronic mRNA.
These results demonstrated that CUG initiation was completely cap-dependent whereas AUG initiation occurred by a mechanism of internal ribosome entry, suggesting the presence of an IRES between the two initiation codons.
Bicistronic mRNAs containing deletions of the first 284, 417, or 495 nucleotides were able to initiate translation at the Mo-MuLV AUG codon (Fig. 3B, lanes 1-8) whereas deletions of the first 521 nt or more abolished translation initiation from the AUG codon (lanes 9-14). However, the ratio of IRES-initiated to cap-initiated translation, that was about 2:1 for the original pHPBiCMC construct (lane 2), changed to 1:1 for pHPBi CMC 284 (lane 4), and to 1:2 for pHPBi CMC 417 and 495 (lanes 6 and 8).
These data enabled us to conclude that Mo-MuLV IRES is contained within a fragment of 126 nt, between positions 495 and 621 of the Mo-MuLV RNA sequence, just upstream from the AUG codon. However, sequences located in 5` of position 495 could influence the IRES efficiency.
In order to analyze the role of these oligopyrimidine tracts, two mutants were generated: either 11 nt of the 600-614 region were mutated to purines (Fig. 4A, CMC m1), or the 573-576 sequence was changed from GUUC to AUGG (CMC m2). COS-7 cells were transfected by the mutated bicistronic vectors (vectors of the series CAT-MuLV-CAT, with or without 5`-hairpin), and expression of the bicistronic mRNAs was analyzed by Western immunoblotting as in Fig. 1B and 3B. The MuLV CAT protein was detected for CMC m1 (lanes 3 and 4) at the same level as the positive control CMC (lanes 1 and 2), whereas it was not visible for CMC m2 (lanes 5 and 6).
These results indicated that the oligopyrimidine tract starting at position 600 was not critical, whereas the 573-576 sequence was required for the IRES function.
Protein factors able to bind the Mo-MuLV IRES were detected
by UV cross-linking assay. A radiolabeled RNA probe containing the IRES
(nt 495-671) was UV irradiated in the presence of a COS-7 S10
cell extract, and analysis of the RNA cross-linked proteins was carried
out by SDS-PAGE (Fig. 5A). The results revealed six
major cross-linked bands migrating at 63, 57, 55, 52, 44, and 38 kDa,
the intensity of which increased with the UV irradiation level (lanes 2 and 3). As expected for proteins, the bands
disappeared upon proteinase K treatment (Fig. 5A, lane 4). The same profile of cross-linked proteins was
observed with a murine NIH-3T3 S10 cell extract (data not shown). The
specificity of these interactions was demonstrated by competition
experiments using an excess of unlabeled Mo-MuLV IRES, EMCV IRES, or
CAT RNAs (20- or 50-fold). Protein cross-linking was competed by
Mo-MuLV IRES (lanes 5 and 6) but not by CAT RNA (lanes 9 and 10), indicating the existence of
specific interactions. Interestingly, the cross-linking of several
proteins was competed by EMCV IRES, particularly a protein migrating at
57 kDa. A cross-linking assay using P-labeled EMCV IRES
probe showed a co-migration of this 57-kDa protein with the PTB
splicing factor (lane 11).
Furthermore, the 57-kDa protein disappeared when the cross-linking assay was performed with a radiolabeled probe corresponding to the m2 inactive mutant of Mo-MuLV IRES (lane 14). These data suggest an involvement of the 57-kDa protein in Mo-MuLV IRES function.
As a complementary experiment, UV cross-linking assays were carried out using labeled EMCV IRES RNA probe, and competition experiments were performed by adding an excess (20-50-fold) of the various unlabeled RNAs described above (Fig. 5B). As expected and according to previous reports(24) , the cross-linking of the major band corresponding to the PTB/p57 splicing factor was competed by EMCV IRES RNA but not by CAT RNA. The cross-linking was partially displaced by Mo-MuLV IRES RNA but not by the m2 mutant IRES RNA. These observations again suggested that the 57-kDa protein interacting with Mo-MuLV IRES and the p57/PTB protein interacting with EMCV IRES could correspond to the same protein, unable to bind to the Mo-MuLV mutant IRES.
S-Labeled PTB was synthesized in vitro in
wheat germ extract (containing no endogenous PTB), and the translation
assays were incubated with biotinylated Mo-MuLV wild type or mutant m2
IRESes. Biotinylated EMCV IRES and U1 RNAs were used as positive and
negative controls, respectively. To check the system, the experiment
was also performed with
S-labeled U1A protein and
biotinylated U1 RNA (Fig. 6, lanes 1-4), as
described in previous reports demonstrating specific interactions with
this methodology(34) . The biotinylated
RNA
[
S]protein complexes were then
precipitated with streptavidine acrylamide beads and analyzed by
SDS-PAGE.
The results showed that the labeled PTB was retained on beads charged with biotinylated Mo-MuLV IRES, as well as with biotinylated EMCV IRES (Fig. 6, lanes 7 and 8). The efficiency of PTB-MuLV RNA interactions was similar to that of PTB-EMCV and U1A-U1 interactions (lane 3). Interestingly, the m2 mutant RNA, shown in Fig. 4to be unable to promote internal initiation, was not able to retain the PTB (lane 9).
In conclusion, these data demonstrate that the PTB protein interacts with the Mo-MuLV IRES and that this interaction is dependent upon the nucleotides 573-576 of Mo-MuLV RNA, the same nucleotides that are required for internal ribosome binding: this favors direct involvement of the PTB in the internal initiation process of Mo-MuLV translation.
These results show that translation of the Moloney murine leukemia virus genomic mRNA occurs by internal ribosome entry and involves the PTB/p57 splicing factor. Moreover we have mapped the IRES within a fragment of 126 nt located between positions 495 and 621 of Mo-MuLV RNA, just upstream of the AUG start codon. We have shown that the PTB-binding site involves nucleotides 573-576, a region required for internal ribosome entry.
MuLV is the first example of a retrovirus whose mRNA translation occurs by internal ribosome entry. Is the mechanism governing this process similar to that of the picornaviruses? The 450 nt long picornavirus IRES presents a pyrimidine-rich tract located 25 nt from its 3`-end and an AUG triplet at the very 3`-end(11) . In a first group, including EMCV, all entering ribosomes initiate at this AUG (36) . In a second group, including foot and mouth disease virus, this AUG is not efficiently used and most of the ribosomes scan to initiate at the next downstream AUG(37) . In a third group, which includes poliovirus, the AUG codon at the 3` of the IRES is not used and all ribosomes scan to the next AUG located 40-160 nt downstream(13, 38) . The 126 nt long Mo-MuLV IRES is shorter than picornavirus IRESes. Similarities do, however, exist between both types of IRESes, particularly an oligopyrimidine stretch found at 21 nt upstream from the MuLV AUG start codon (Fig. 4). Mo-MuLV IRES would then be related to the first group with translation initiation occurring at the AUG located at the IRES 3`-end. The oligopyrimidine tract at position -21 is not required, however, whereas a UUCU motif at position -45, corresponding to the PTB protein-binding site, is necessary ( Fig. 4and 6). These data are consistent with recent studies showing that the picornavirus oligopyrimidine tract at position -25 is not necessary as a nucleotide sequence, in the cases of EMCV and of another cardiovirus, the Theiler's murine encephalomyelitis virus(39, 40) . The relevant parameter for EMCV could be the distance of the AUG triplet from upstream IRES elements. For Theiler's murine encephalomyelitis virus, both oligopyrimidine tract and AUG at a fixed distance seem dispensable: initiation occurs in a cis-acting element, the starting window, wherefrom the IRES-bound ribosome becomes ready to scan and initiate.
The similarity between Mo-MuLV and EMCV IRESes has been confirmed by the demonstration that the same trans-acting factor, p57/PTB, interacts with both IRESes ( Fig. 5and Fig. 6). The inability of the PTB protein to interact with the m2 mutant IRES strongly suggests that its binding site involves the oligopyrimidine tract located around position -45. This oligopyrimidine tract belongs to a stem-loop homologous to the end of EMCV stem-loop E required for the binding of PTB in EMCV RNA, located at position -405(24) . The correlation between the absence of PTB binding and of internal ribosome entry (Fig. 4) suggests a direct role of the PTB in this process. This is in agreement with previous reports demonstrating, by PTB-depletion of HeLa or ascites cell extracts with anti-PTB antibodies or competitor RNAs, that PTB has an essential role in EMCV translation by internal ribosome entry(30, 41) .
The cross-linking patterns of
Mo-MuLV and EMCV IRESes are very different: PTB is the major protein
bound to EMCV IRES, whereas at least six proteins are bound to Mo-MuLV
IRES. The band migrating at 52 kDa might correspond to La antigen;
however, the other proteins have never been described and may be
specific to Mo-MuLV translation initiation. We hypothesize that in the
case of Mo-MuLV, the ribonucleoprotein responsible for IRES function,
termed the IRESome (42) , would contain several components in
addition to PTB. This has already been suggested for EMCV IRES in
studies showing that PTB by itself was not sufficient to promote the
formation of the active IRESome(30, 41, 43) .
Consistent with this hypothesis, several other RNA-binding proteins,
that do not cross-link to the complete EMCV IRES, are able to
cross-link to IRES fragments(42) . The IRESome could also
involve protein-protein interactions that cannot be revealed by UV
cross-linking. The implication of several IRESome components has been
observed for Theiler's murine encephalomyelitis virus, human
rhinovirus, and hepatitis C virus(44) . ()Furthermore, in the case of poliovirus, an active
multiprotein complex of 240 kDa including PTB and La protein has been
purified(43) . Interestingly, the p63, p44, and p38 factors
interacting with Mo-MuLV IRES seem to be different from factors
interacting with picornavirus IRESes (at least EMCV IRES), suggesting
the existence of different types of IRESomes (Fig. 5).
What is the role of the internal ribosome entry process when the Mo-MuLV mRNA is capped and can be translated by the classical cap-dependent scanning mechanism, as shown in case of the CUG codon? Two nonexclusive hypotheses can be proposed for the role of the IRES.
First, the most evident function of Mo-MuLV IRES is to discriminate between the two alternative initiation codons: the AUG is used following internal ribosome entry whereas the CUG is translated in a cap-dependent manner. This process allows control by trans-acting factors (including PTB) of the relative synthesis of the two gag precursors that have different localizations and functions. Thus, in the case of Mo-MuLV retrovirus the process of internal entry governs alternative initiation of translation.
Second, the IRES may be a cis-acting translational enhancer, whose activity would be trans-controlled by IRESome factors. Internal ribosome binding bypasses the repression of translation initiation that is expected from the scanning of a long and structured leader: in that context internal initiation corresponds to a new mechanism of translational activation or derepression.
The IRES role of translational enhancer could also concern other retroviruses species devoid of an alternative initiation system but having in all cases a long 5`-untranslated region. Such a process could help the virus to resist the cellular mechanisms directed against its expression or allow it to be expressed in conditions of low cap-dependent translation, for instance, cell growth arrest induced by HIV-1.
What is the importance of the IRES for viral replication? The shift of retroviral genomic RNA from translated mRNA to encapsidated genome seems to result from RNA conformational changes, involving RNA dimerization(2, 45, 46) . The passage from monomer to dimer conformation might simultaneously inactivate the IRES and activate the packaging sequence, resulting in translation blockade and genome packaging(47, 48, 49) . The fate of retroviral mRNA could be governed by the balance between both activities, controlled by internal initiation factors (translational activators) and by nucleocapsid protein (promoting RNA dimerization and encapsidation).
We have shown that IRES of Mo-MuLV is 126 nt long, i.e. much shorter than the 450 nt long picornavirus IRESes. This gives us new perspectives for the construction of a new generation of bicistronic vectors containing Mo-MuLV IRES used for co-expression of two (or more) proteins. Mo-MuLV IRES is of special interest with regard to the design of retroviral vectors for gene therapy. Consequently, the Mo-MuLV IRES should provide biotechnological applications.
Finally, the identification of an IRES in Mo-MuLV mRNA
participates in the emergence of a new concept for translation
initiation of eukaryotic mRNAs, as an increasing number of cellular
mRNAs contain an
IRES(26, 27, 28, 29) . ()Such mRNAs possess long untranslated regions and are poor
candidates for the scanning mechanism, thus favoring a role of the IRES
as translational enhancer controlled by cell specific trans-acting factors. Internal initiation of translation could
generalize to messengers with long leader sequences, mostly encoding
proteins whose level of expression is crucial to cell life, such as
growth factors and transcriptional regulators.