©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternative Translation Initiation of the Moloney Murine Leukemia Virus mRNA Controlled by Internal Ribosome Entry Involving the p57/PTB Splicing Factor (*)

(Received for publication, April 14, 1995; and in revised form, June 12, 1995)

Stéphan Vagner (1)(§) Axel Waysbort (1) Marc Marenda (1) Marie-Claire Gensac (1) François Amalric (2) Anne-Catherine Prats (1)(¶)

From the  (1)From INSERM U397, Endocrinologie et Communication Cellulaire, Institut Louis Bugnard, C. H. U. Rangueil, Avenue Jean Poulhès, 31054 Toulouse cedex and the (2)Laboratoire de Biologie Moléculaire des Eucaryotes du CNRS, 118, route de Narbonne, 31062 Toulouse cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)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. (^2)

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, (^3)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.


MATERIALS AND METHODS

Plasmid Construction

pMC, pBi CMC, and pHPBi CMC (Fig. 1)

The fusion of CAT coding sequence with the 5` of Mo-MuLV was performed by PCR, using the 5`- and 3`-primers 5`-AAATCTAGAGCGCCAGTCCTCCGA-3` and 5`-AAAGGCGCCATCTTTCCAGTCACC-3`, respectively, and the template pMLVAC-7 (4) . The resulting fragment corresponded to nt 1-671 of the Mo-MuLV sequence, with a 5`-XbaI site and a 3`-NarI site. This fragment was cloned into the XbaI and NarI sites of the mono- and bicistronic vectors pFC1, pBI-FC1, and pHP-FC1 previously described, replacing the FGF-2 leader sequence(31) .


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 (DeltaG = -40 kCal/mol). pMC HP1 and pMC HP284, derived from pMC, with addition of a very stable hairpin (DeltaG = -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.



pMC HP1 and pMC HP284 (Fig. 1)

Insertion of a hairpin of DeltaG = -80 kCal/mol into vector pMC was performed by insertion of the double-stranded (autocomplementary oligonucleotide) 5`-CTAGACTCGAGGCGAGGTGGCGACCGCGCATGCGCGGTCGCCACCTCGCCTCGAGT-3` either into the pMC XbaI site located at the 5`-end or into the pMC SpeI site located at position 284 of the Mo-MuLV leader.

pBi CMC 284 and pBi CMC 565 (Fig. 3)

Deletions of Mo-MuLV sequence 1-284 and 30-565 in the bicistronic vectors were obtained by enzymatic digestion of the vectors pBi CMC or pHPBi CMC with XbaI plus SpeI (pBi CMC 284), or SmaI plus PstI (pBi CMC 565), followed by Klenow treatment and ligation.


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.



pBi CMC 417, pBi CMC 495, pBi CMC 521, or pBi CMC 543 (Fig. 3)

Four PCR fragments were synthesized, using four 5`-primers 5`-CCCTCTAGACCCGATCGTTTTGGACTCTTTGG-3`, 5`-CCCTCTAGACAGTTCCCGCCTCCGTCTG-3`, 5`-CCCTCTAGACGTTTCGGTTTGGGACCG-3`, or 5`-CCCTCTAGACGCGCCGCGCGTCTTGTC-3`, corresponding to Mo-MuLV sequences 417-439, 495-514, 521-538, and 543-561, respectively, with a 5`-XbaI site, and one 3`-primer 5`-TTTGAGCTCAGATCTCATTACGCCCCGCCCTGCCA-3` complementary to the 3` region of CAT with a SacI site. The four PCR fragments were cloned into the XbaI and SacI sites of pBi CMC (replacing Mo-MuLV 5` and CAT sequences).

pBi CMC m1, -m2, pHPBi CMC m1, -m2 (Fig. 4)

Mutagenesis of the IRES was carried out by generating two PCR fragments: the 5`-primers were 5`-CTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGAGAGAGTAAAAGAGAGAGAATATGGGCC-3` and 5`-CTGCTGCAGCATCATGGTGTGTTGTCTCTG-3`, corresponding to nt 562-627 and nt 562-584, with mutations of nt 600-614 and 573-576, respectively, containing a 5`-PstI site. The 3`-primer was complementary to CAT 3`-end with a SacI site (see above). The two PCR fragments were cloned into the PstI and SacI of pBi CMC and pBiHP CMC, replacing the corresponding wild type fragment (PstI site at position 565 of Mo-MuLV leader sequence and SacI site in 3` of CAT). This resulted in pBi CMC m1 and m2, and in pBiHP CMC m1 and m2 (Fig. 4).


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.



pMC-495 and pMC-m2

The monocistronic version of the mutant Delta1-495 and m2, used for in vitro synthesis of RNA fragments, resulted from introduction of the XbaI-SacI FGF-CAT fragment of pBi CMC 495 and -m2 into the pSCT vector (downstream of T7 promoter).

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 U1Delta RNAs were a gift from I. W. Mattaj.

DNAs were checked by sequencing with the dideoxy method.

In Vitro Transcription and Translation

The transcription templates corresponded to various linearized DNAs. pMC, pMC m2, pMC 495-621, and pMC 495-621 m2 allowed synthesis of the Mo-MuLV RNA fragments 1-671 and 495-671 ( Fig. 5and Fig. 6). pTM1 allowed synthesis of the EMCV RNA fragment 261-837(24) , pSCT CAT synthesis of the CAT RNA(31) , and pKS-PTB synthesis of the PTB mRNA. Uncapped RNAs were generated in vitro by T7 or T3 RNA polymerase according to the manufacturer's instructions. RNA transcripts were quantitated by absorbance at 260 nm and ethidium bromide staining on agarose gel and their integrity verified. RNA labeling was performed in 50 µl in the presence of 60 µCi of [P]CTP (with 30 µM of unlabeled UTP). RNA biotinylation was performed with 0.5 mM of biotine-UTP (Clontech).


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 10^5 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^2 at 254 nm, except for lanes 2 (A and B) corresponding to an irradiation at 200,000 µJ/cm^2. 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^2, 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 RNAbullet[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), U1DeltaB (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).

COS-7 Cells Transfection

COS-7 monkey cells were transfected by the DEAE-dextran method, as described previously(31) . 1 µg/ml of each plasmid was incubated with the cells for 20 min at 37 °C in the presence of 1 mg/ml DEAE-dextran, then chloroquine was added at 40 µg/ml and the incubation continued for 4 h. The DNA was then removed and the cells incubated in 10% dimethyl sulfoxide for 2 min. Cell lysates were prepared 48 h later.

Cellular RNA Purification and Northern Blotting

Total cellular RNA was prepared by the Trizol method (Life Technologies), as described previously, from pellets containing 5 10^6 transfected scraped cells. To eliminate any DNA contamination, the RNA was treated with 10 units of RNase-free DNase I for 30 min at 37 °C, then treated for 15 min with proteinase K at 100 µg/ml.

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 and CAT Assays

COS-7 cell lysates were prepared 48 h post-transfection by scraping cell monolayers. Cell pellets were frozen-thawed, resuspended in 0.1 M Tris, pH 7.8, and sonicated. Total proteins were quantified by Bio-Rad assay (absorbance at 595 nm), and 3 µg of proteins from each cell lysate were used for Western immunoblotting.

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) .

UV Cross-linking Assays

S10 cytoplasmic extracts from COS-7 cells were prepared as already described(32) . Subconfluent cell monolayers were scraped in 100 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5, and centrifuged; the cell pellet was resuspended in 10 mM NaCl, 10 mM Tris-HCl, pH 7.5, and frozen and thawed three times. The extract was centrifuged at 10,000 g for 5 min and the supernatant (S10) brought to 5% (v/v) glycerol and frozen in aliquots at -80 °C.

For UV cross-linking experiments, 1.10^5 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(2), 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, 10^5 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^2 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.

Streptavidine Acrylamide Precipitation of PTB/p57 Protein with Biotinylated RNA

This experiment was performed as described(34) . 1 µl of biotinylated RNA (200 ng/µl in solution containing 2 mg/ml of calf liver tRNA (Boehringer Mannheim) was incubated with 1 µl of a S-labeled PTB protein (translated in wheat germ extract) in 8 µl of KHN buffer (150 mM KCl, 20 mM HEPES, pH 7.9, 0.05% Nonidet P-40, 0.2 mM dithiothreitol) for 1 h at 25 °C. The mixture was then diluted with 500 µl of KHN buffer, transferred to a tube containing 30 µl of streptavidine acrylamide beads (Pierce), and incubated 1 h at 20 °C. The beads were collected by centrifugation, washed four times with 1 ml of KHN buffer, resuspended in 10 µl of sample buffer, and boiled for 5 min. After centrifugation, the supernatant was loaded on to an SDS-PAGE gel. The gel was exposed after electrophoresis and after a fluorography treatment using Amplify (Amersham).


RESULTS

An Internal Ribosome Entry Site Is Present in the Mo-MuLV RNA Leader

We looked for an IRES in the Mo-MuLV RNA leader by using two different strategies to block cap-dependent initiation of translation.

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 (DeltaG = -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 (DeltaG = -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.

The IRES Is Located between the CUG and AUG Codons within a Fragment of 126 nt

To define the minimal sequences of Mo-MuLV IRES, we first generated a series of 5` deletions of the 5`-untranslated region in a bicistronic vector with or without a hairpin structure (DeltaG = -40 kCal/mol) at the 5`-end (Fig. 3A). The constructs were transfected in COS-7 cells and translation products were analyzed by Western immunoblotting as in Fig. 1B.

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.

Role of Oligopyrimidine Tracts in the Internal Entry Process

It has been shown for several picornaviruses that the IRES involves an oligopyrimidine tract, starting with a UUUUC or UUUC motif, located some 25 nt upstream of the start codon(11, 12, 13) . We therefore looked for a similar possibility in the Mo-MuLV IRES. The RNA sequence located upstream from AUG-621 is pyrimidine-rich and there are, in particular, two oligopyrimidine tracts UUUCU and UUCU at positions 600 and 574, respectively. As previously reported, these oligopyrimidine tracts are involved in two hairpin structures (35, Fig. 4B).

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.

Characterization of IRES Binding Factors

Identification of the PTB/p57 splicing factor and La autoantigen involved in the internal entry process of EMCV and poliovirus (24, 25) prompted us to look for trans-acting factors mediating this same phenomenon in Mo-MulV.

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.

Specific Interactions of the Splicing Factor PTB/p57 with the Mo-MuLV IRES

To clearly demonstrate the existence of interactions between the PTB protein and the Mo-MuLV IRES, we used streptavidine acrylamide beads to trap complexes of in vitro translated S-labeled protein with biotinylated RNA.

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 RNAbullet[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.


DISCUSSION

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) . (^4)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) . (^5)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.


FOOTNOTES

*
This work was supported by grants from the Association pour la Recherche contre le Cancer and the Conseil Régional Midi-Pyrénées. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Association pour la Recherche contre le Cancer.

To whom correspondence should be addressed: Tel.: 33-61322142; Fax: 33-61322141.

(^1)
The abbreviations used are: MuLV, murine leukemia virus; Mo-MuLV, Moloney MuLV; IRES, internal ribosome entry site; EMCV, encephalomyocarditis virus; FGF, fibroblast growth factor; nt, nucleotide(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis.

(^2)
S. Vagner, C. Touriol, M.-C. Gensac, F. Amalric, F. Bayard, H. Prats, and A.-C. Prats, manuscript in preparation.

(^3)
A.-C. Prats, unpublished results.

(^4)
R. Jackson, personal communication.

(^5)
S. Vagner and A.-C. Prats, unpublished results.


ACKNOWLEDGEMENTS

We thank S. Audigier for correction and criticism of the paper. We also thank F. Bayard, J. C. Faye, and H. Prats for helpful discussions and D. Warwick for English proofreading. We thank P. Schonberger Mc Caw and P. A. Sharp for the gift of PTB cDNA, and I. W. Mattaj for the gift of U1A, U1, and U1DeltaB cDNAs.


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