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Originally published In Press as doi:10.1074/jbc.M608750200 on November 8, 2006

J. Biol. Chem., Vol. 282, Issue 1, 132-141, January 5, 2007
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Picornavirus Internal Ribosome Entry Site Elements Can Stimulate Translation of Upstream Genes*

Christiane Jünemann1, Yutong Song, Gergis Bassili, Dagmar Goergen, Jura Henke, and Michael Niepmann2

From the Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany

Received for publication, September 11, 2006 , and in revised form, November 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K+ concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K+ concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m7GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Internal ribosome entry site (IRES)3 elements are cis-acting RNA regions that confer the internal entry of ribosomes to the RNA translation start site independent of the process of capdependent scanning from the 5'-end of the RNA (1, 2). Such IRES elements were first discovered in picornaviruses like encephalomyocarditis virus (EMCV) (3), poliovirus (4), and foot-and-mouth disease virus (FMDV) (5). Also the translation of hepatitis C virus (HCV) (6) and of several cellular mRNAs is driven by IRES elements (2).

The picornaviral IRES elements are classified in three groups according to their sequences and secondary structures, the type I elements of the entero-/rhinovirus group (including poliovirus), the type II elements of the cardio-/aphthovirus group (including EMCV and FMDV), and the type III element of hepatitis A virus (1). After infection of a susceptible cell, these IRES elements guide the small ribosomal subunit to an AUG triplet in a starting window at the 3'-border of the IRES (1, 2, 7). Ribosome binding to the picornavirus IRES elements is mediated by a number of cellular RNA-binding proteins (8) that fall into two groups. On one hand, all standard eukaryotic translation initiation factors (eIFs) are required (9), except the actual cap-binding protein eIF4E, a protein that associates with the large adaptor protein eIF4G (and the RNA-helicase eIF4A) in the heterotrimeric cap-binding complex called eIF4F and promotes the translation initiation of capped mRNAs. The initiation factors bind to a large Y-shaped RNA structure in the IRES of FMDV and EMCV (arrows in Figs. 1A and 5A) (10–16) or to a bulged domain in the poliovirus IRES (arrows in Fig. 5A) (17, 18) and facilitate ribosome entry downstream of the IRES. On the other hand, picornavirus IRES RNAs recruit a variety of cellular RNA-binding proteins that are usually not involved in translation, like the La protein, poly(rC)-binding protein 2, and the polypyrimidine tract-binding protein PTB (8), to stimulate their IRES-mediated translation. In contrast to the picornaviruses, the HCV IRES is able to bind to the ribosome in the absence of virtually all initiation factors or accessory proteins (19, 20).

Picornavirus IRES elements are widely used in molecular biology and its applications. In gene therapy, IRES elements are used in dicistronic or multicistronic vectors to drive multigene expression from single mRNAs, e.g. a therapeutic gene together with a selectable marker or two subunits of a heterodimeric protein (21–26). In artificial virus replicon systems, the internal insertion of an additional IRES element (most commonly the EMCV IRES) is widely used to uncouple the required constitutive expression of the viral nonstructural replication proteins from the biologic activity of the viral cis-signals under investigation (27–31). In dicistronic mRNA systems, which are commonly used to investigate IRES function on its own, the expression of the upstream gene usually serves as an internal control for standardizing assays that measure IRES activity monitored by downstream reporter expression (3, 4, 32–34) (see also Fig. 1A).

Although picornavirus IRES elements are known to be active at high concentrations of monovalent cations (35, 36), translation of most cellular mRNAs is usually optimal at salt concentrations lower than 80 mM (37–39). Using dicistronic mRNA systems for analyzing the activity of mutant IRES elements of FMDV (16) and poliovirus, we made a surprising observation: the first reporter gene in such dicistronic mRNAs was very actively translated also at high salt concentrations in the presence of a functional downstream IRES, although the same reporter gene was translated with poor efficiency at high salt concentrations when placed in a monocistronic mRNA.

Investigating this effect in detail, we show in this study that a functional picornavirus IRES in a dicistronic mRNA may support the activity not only of the downstream but also of the upstream reporter gene at high salt concentrations in cis. Analysis of different experimental parameters influencing this effect shows that the enhanced availability of the initiation factor eIF4F provided by a functional picornavirus IRES on the same RNA molecule in cis causes this translation enhancement effect. This effect may have important implications mainly for the investigation of the effect of mutations on translation and viral RNA replication in dicistronic replicon systems as well as on the analysis of IRES function on its own.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The dicistronic expression vector pCAT-FM was renamed for clarity from pD12 (13) and contains downstream of an SP6 polymerase promoter the chloramphenicol acetyltransferase (CAT) gene and the FMDV IRES with the 11th AUG of FMDV fused to the firefly luciferase (Fluc) gene, followed by SV40 tAg splice and p(A) signals to allow efficient processing of transcripts after transfection. Plasmids pCAT-FM-up-CG and pCAT-FM-up-4 are pD12 derivatives described previously (16). pCAT-FM-d3 was derived from pD12 by exactly deleting FMDV IRES stem-loop 3 sequences. pFM is renamed from pM12 (13) for clarity; it contains the FMDV IRES and the firefly luciferase gene. pMPolio (17) contains downstream of an SP6 polymerase promoter the complete poliovirus type 1 (Mahoney) 5'-UTR sequence with the authentic initiator AUG fused to the Fluc coding sequence. For construction of pRL-FM-wt, pRL-FM-up-CG, and pRL-FM-up-4, the CAT reporter gene was removed from the respective pCAT-FM-plasmid, and the T7 polymerase promoter sequence together with the Renilla luciferase (Rluc) gene from phRL-null (Promega) were inserted. For construction of the dicistronic expression vector pRL-PV-wt, the Rluc gene from phRL-null was inserted upstream of the poliovirus IRES in pMPolio. pRL-PV-S.3 and pRL-PV-{Delta}V were generated by replacing the wild-type poliovirus IRES in pRL-PV-wt by the mutated poliovirus IRES elements from plasmids pMPolio-S3 and pMPolio-{Delta}V (18). For construction of pCAT-PV-wt, the CAT gene from pCAT-FM was ligated into pRL-PV-wt, replacing the Rluc gene. pCAT-PV-S.3 and pCAT-PV-{Delta}V were generated by replacing the wt poliovirus IRES in pCAT-PV-wt by the mutated poliovirus IRES from plasmids pMPolio-S3 and pMPolio-{Delta}V (18). pHPI933 (40) was kindly provided by P. Mavromara (Athens, Greece). In pHPI933-d3abc, HCV IRES nucleotides 160–249 (corresponding to stem-loop IIIa-c sequences) were deleted using AgeI and NheI. pFMDV14 contains the FMDV sequence downstream of an SP6 promoter (15). pcDNA3.1(+)/CAT was purchased from Invitrogen. pcDNA4/HisMax/lacZ (Invitrogen) was used for co-transfection controls.

In Vitro Transcription—Plasmids were usually linearized downstream of the Fluc gene. RNAs were synthesized in vitro at 37 °C for 60 min by using T7 or SP6 RNA polymerase, respectively. For synthesis of capped RNAs, the rGTP concentration was reduced to 50 µM, and m7GpppG cap analogue (Roche Applied Science) was added to a concentration of 500 µM. The capped RNA was purified by precipitation with 2.5 M ammonium acetate, polyadenylated using poly(A) polymerase (USB Corp., Cleveland, OH), and again purified by precipitation with 2.5 M ammonium acetate. RNA for translation of FMDV L protease was transcribed from pFMDV14 (15). The concentrations of all transcripts were determined photometrically and by agarose gel electrophoresis.

In Vitro Translation—Translation reactions were performed in nuclease-treated rabbit reticulocyte lysate (RRL; Promega) or in non-nuclease-treated cytoplasmic HeLa extract (41). 10-µl reactions usually contained 200 ng of RNA and 4.4 µl of extract. For labeling of proteins, 0.2 µl of [35S]methionine (15 mCi/ml; Amersham Biosciences) was included. The RRL contained 113 mM of endogenous K+-acetate added by the supplier. To the translation reactions, KCl was supplemented to total concentrations of up to 160 mM as indicated. m7GDP or GDP was supplemented to 100 µM were indicated. The proteins were separated by SDS-polyacrylamide gel electrophoresis, and gels were analyzed by autoradiography. The gels were checked for equal sample loading if appropriate. m7GDP, a eIF4E inhibitor, was used at 100 µM concentration. Hippuristanol, an inhibitor of eIF4F activity, was kindly provided by J. Tanaka and J. Pelletier (42) and used at 10 µM concentration.

Cell Culture and Transfection—HeLa and BHK21 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% (v/v) fetal bovine serum, L-glucose (4500 mg/liter), L-glutamine, and 1% (v/v) penicillin-streptomycin and were incubated at 37 °C in a humidified atmosphere with 5% CO2. RNA transfections were performed with the Transmessenger reagent (Qiagen) according to protocols provided by the supplier.

Luciferase Assays—Renilla and firefly luciferase (Promega) as well as beta-galactosidase reporter activity (Roche Applied Science) activities were determined essentially according to protocols provided by the reagent suppliers. Activities of reporter expression were usually measured 4 h after transfection.

Western Blots–eIF4G and its C-terminal cleavage products were detected using an anti-eIF4G antiserum (BD Transduction Laboratories) that recognizes the C terminus of eIF4G (15).

Northern Blots—RNAs were detected with a 32P-labeled RNA probe complementary to the 3'-region of the firefly luciferase gene (43) using standard procedures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dicistronic mRNAs are widely used in IRES research to monitor the activity of an IRES or its mutant variants. In such experiments, the efficiency of translation of the second reporter gene, which is located downstream of the IRES, serves as a measure for the biologic activity of the IRES. At the same time, expression of the upstream first reporter gene of the dicistronic mRNA is usually used as an internal standard to level out differences between the different reactions of a given experiment that are due to slight variations in cell density, transfection efficiency, and other parameters of the experiment in question.


Figure 1
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  		FIGURE 1.
A picornavirus IRES enhances first reporter translation in a dicistronic mRNA. A, the dicistronic expression cassette with the CAT gene as first reporter, the FMDV IRES with its five stem-loops, and the Fluc gene as second reporter. The RNA was in vitro transcribed from an SP6 promoter. The arrow indicates the site of the up-4 mutation. B, the up-4 mutation in the FMDV IRES stem-loop 4. C, 35S-labeled proteins from in vitro translations in RRL at 50 and 120 mM potassium (K+) using the uncapped and nonpolyadenylated dicistronic mRNAs as indicated. D, enzymatic activities measured after in vitro translations as in C. Expression values (in percentages of activity, % act.) are shown for first reporter CAT (black bars, C) and second reporter Fluc (gray bars, F). E, Northern blots with RNAs re-extracted from in vitro translations as in C, analyzed with an antisense Fluc probe (43).

 
A Picornavirus IRES Can Enhance Translation of an Upstream Reporter Gene at Physiological Salt Concentrations–In the course of experiments designed to analyze the effects of mutations in the eIF4G-binding site in the FMDV IRES (16), we made a surprising observation when we used dicistronic mRNAs (Fig. 1A) to compare the translation efficiency of the up-4 mutant IRES (Fig. 1B) with wild-type IRES activity in the translation-competent RRL. These dicistronic mRNAs contain CAT as first reporter, the FMDV IRES, and the firefly luciferase (Fluc) as second, IRES-dependent reporter. At a potassium concentration of 50 mM, Fluc expression was significantly decreased with the IRES mutant up-4, whereas expression of the first reporter CAT was essentially unchanged and could be regarded as an internal standard (Fig. 1, C, compare lane 2 with lane 1, and D, 50 mM, black bars). When the same dicistronic mRNAs were translated in the presence of 120 mM K+, which is actually close to the physiological K+ concentration in living cells, we observed that the expression of the IRES-dependent Fluc protein was seriously impaired with the up-4 mutant IRES as expected (16). However, to our surprise also the level of CAT expression from the dicistronic mRNA containing the mutant IRES was much lower than CAT expression from the mRNA with the wild-type IRES (Fig. 1, C, compare lane 4 with lane 3, and D). Northern blots (Fig. 1E) show that the differences in expression were not due to differences in RNA amounts or stability.

Thus, the presence of an active IRES in the dicistronic mRNA not only drives expression of the downstream reporter gene (which is regarded as a measure for IRES activity) but also positively influences first reporter expression, which is inefficient at elevated salt concentrations in the absence of an functional IRES (see also below).

In the following, we performed a series of experiments to answer these questions: (i) Does the translation enhancement depend on the biological activity of the IRES or just on IRES RNA structure? (ii) Is the translation enhancement dependent on the type of picornavirus IRES? (iii) Does the effect depend on a special feature of a particular first reporter gene? (iv) Does the translation enhancement occur preferentially in cis on the same molecule of RNA or does it also occur in trans between different RNA molecules? (v) Is a second reporter gene translation required? (vi) Which factors may be involved in the translation enhancement?

A Functional IRES Is Required for Translation Enhancement–To answer the above questions, we first analyzed the translation efficiency of the CAT first reporter gene contained in the dicistronic RNA at different K+ concentrations in comparison with the CAT gene contained in a monocistronic mRNA (Fig. 2A). With the dicistronic mRNA bearing the FMDV IRES, IRES-dependent Fluc expression was very efficient up to K+ concentrations of 120 mM, still significantly efficient at 130–140 mM but inefficient only above 140 mM (Fig. 2B). Expression of the CAT gene contained in the same mRNA was efficient up to K+ concentrations of 120 mM but declined at K+ concentrations of 130 mM or more. To check equal sample loading, the gels were Coomassie-stained prior to x-ray film exposure (not shown), and experiments performed with different RNA concentrations revealed that the effect was not concentration-dependent (not shown).

In contrast, expression of the CAT gene from a monocistronic mRNA (Fig. 2D) showed a sharp decline at K+ concentrations above 70 mM and was nearly undetectable at K+ concentrations of 100 mM or higher. Thus, CAT first reporter translation was enhanced significantly in the presence of the downstream FMDV IRES at elevated salt concentrations.

The Translation Enhancement Is Independent of the Type of Picornavirus IRES—To find out whether the observed translation enhancement is an effect special to a certain IRES structure, we used the poliovirus IRES instead of the FMDV IRES in a dicistronic mRNA. In this case, the expression of Fluc driven by the poliovirus IRES was not as efficient at higher salt concentrations (Fig. 2C) as with the FMDV IRES, consistent with the finding that the poliovirus IRES has a salt optimum for translation at about 80 mM K+ instead of the 120 mM K+ optimal for FMDV IRES activity in vitro (36). However, also with the poliovirus IRES, first reporter CAT expression was significantly more efficient in the range of 80–100 mM K+ (Fig. 2C) compared with CAT expressed from the monocistronic mRNA (Fig. 2D). In conclusion, the enhancement of first reporter gene translation in a dicistronic mRNA depends on the presence of a functional IRES rather than on any particular RNA sequences or secondary structures unique to a certain IRES element.


Figure 2
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  		FIGURE 2.
Efficiency of reporter gene translation at different salt concentrations. A, expression cassettes used in B–D. Dicistronic RNAs contain either the wt FMDV IRES (pCAT-FM) or the wt poliovirus IRES (pCAT-PV). As a control, the CAT gene was used in a monocistronic RNA in vitro transcribed from plasmid pcDNA3.1(+)/CAT. B, 35S-labeled CAT and Fluc proteins from in vitro translations with uncapped dicistronic pCAT-FM RNA in RRL at different K+ concentrations. C, translation of Fluc and CAT proteins from dicistronic pCAT-PV RNA. D, translation of CAT protein from monocistronic CAT RNA. E, expression cassettes used in F–H. Dicistronic RNAs are as in A but contain the Rluc as first reporter. As a control, the Rluc gene was used in a monocistronic RNA in vitro transcribed from phRL-null. F, 35S-labeled Fluc and Rluc proteins from in vitro translations with dicistronic pRL-FM RNA in RRL at different K+ concentrations. G, translation of Fluc and Rluc proteins from dicistronic pRL-PV RNA. H, translation of Rluc protein from monocistronic phRL-null RNA.

 
The Translation Enhancement Is Independent of the Nature of the First Reporter Gene—When we exchanged the first reporter gene CAT for the unrelated Renilla luciferase (Rluc) gene (for the constructs, see Fig. 2E), we found that Rluc expression at elevated K+ concentrations was stronger in the presence of the poliovirus IRES in the same mRNA (Fig. 2G) compared with Rluc present only in a monocistronic mRNA (Fig. 2H) and even stronger in the presence of the FMDV IRES (e.g. compare lane 6 in Fig. 2, F–H). Thus, the enhancement of the efficiency of translation of the first reporter gene in a dicistronic mRNA is independent of the nature of the first reporter gene.

Translation in HeLa Extract—To confirm that the results obtained by in vitro translations using reticulocyte lysate reflect biologically relevant phenomena, we also used cytoplasmic HeLa extract, which allows translation in an environment close to normal cellular conditions (41) (Fig. 3).

With this extract, we observed that Renilla luciferase expression from a monocistronic mRNA slightly differs from the expression in RRL by showing maximal translation at higher salt concentrations. However, the overall efficiency of Rluc translation from the monocistronic mRNA was low. In contrast, when the Rluc gene was placed in the dicistronic mRNA together with the downstream FMDV IRES driving Fluc expression (pRL-FM RNA) (Fig. 3A), the expression of the first reporter Rluc was enhanced enormously in a range from 90 to 120 mM K+, thereby exhibiting a salt optimum at slightly lower concentrations than the second reporter Fluc gene, as was also observed with RRL. Also with the poliovirus IRES (Fig. 3B), translation of the Rluc gene was strikingly enhanced when placed in the dicistronic mRNA compared with the monocistronic mRNA, correlating with the optimum for the poliovirus IRES-driven second reporter Fluc expression. These results confirm that also in HeLa extract, first reporter expression can be considerably stimulated by a downstream IRES.

The Translation Enhancement Acts in cis—The stimulating effect of the picornavirus IRES elements on upstream reporter translation was found in dicistronic mRNAs, suggesting that the effect occurs in cis. To confirm that the translation enhancement actually works in cis but not in trans, we performed in vitro translation reactions at different salt concentrations in which the two different reporter genes were provided by two separate mRNAs, one mRNA with the FMDV IRES driving Fluc translation, and the other mRNA with the Rluc gene (Fig. 4A). The result shows that the Fluc reporter protein under control of the FMDV IRES is translated well at high K+ concentrations like from a dicistronic mRNA (Fig. 2F), whereas the Rluc protein is expressed well only at very low salt concentrations up to 60 or 70 mM. This is in contrast to the previous observation that Rluc was expressed efficiently at K+ concentrations of up to 100 mM and more when the gene was placed as first cistron in a dicistronic mRNA (compare Fig. 2F). Thus, the translation enhancement effect acts in cis on the same molecule of RNA but not in trans.


Figure 3
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  		FIGURE 3.
Reporter gene translation at different salt concentrations in HeLa extract. A, in vitro translation of dicistronic (dic.) mRNA with the FMDV IRES in HeLa extracts. Enzymatic activities are given in relative light units (RLU). Solid line, first reporter Rluc activity expressed from dicistronic pRL-FM RNA (see Fig. 2E); dotted line, second reporter Fluc activity expressed from dicistronic pRL-FM; dashed line, Rluc activity expressed from monocistronic phRL-null. B, in vitro translation of dicistronic mRNA with the poliovirus IRES in HeLa extracts. The lines are assigned as for A.

 


Figure 4
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  		FIGURE 4.
Translation is not enhanced in trans. A, monocistronic mRNA with the wild-type FMDV IRES driving Fluc expression was in vitro transcribed from plasmid pFM. The second mRNA, in vitro transcribed from phRL-null, contains the Rluc gene. B, 35S-labeled Fluc and Rluc proteins from in vitro translations with RNAs from pFM and phRL-null in RRL at different K+ concentrations.

 
The Extent of Translation Enhancement Correlates with IRES Function—To confirm that translation enhancement depends on IRES function, we used the FMDV and the poliovirus IRES together with a set of IRES mutations that impair IRES activity (16, 18) (Fig. 5, A–C).

In the FMDV IRES, the up-4 mutation also used above not only exchanges four nucleotides of the primary sequence but also alters the RNA secondary structure in the eIF4G-binding site of the IRES (Fig. 5B). In contrast, the up-CG mutation just exchanges the sequence of two nucleotides extruding from the double-stranded RNA but does not alter RNA secondary structure. Thus, an effect of this mutation on first or second reporter activity must be assigned to a change in the biological activity of the IRES but not to a mere change in IRES RNA secondary structure. When this up-CG IRES variant was used in comparison with the wt FMDV IRES and the up-4 mutant (Fig. 5, D and E), it became evident that also this mutation affected first reporter expression, both with CAT (Fig. 5E, compare lane 3 with lane 1) and Renilla luciferase (Fig. 5E, compare lane 6 with lane 4) as first reporter gene. Northern blots (Fig. 5F) show that the differences in expression are not due to differences in RNA amounts or stability.

Moreover, we also used mutations in the poliovirus IRES. Either the entire stem-loop V that bears the binding sites for the canonical initiation factors (18) was deleted (mutant {Delta}V), or a point mutation exchanging a C to a U like in the poliovirus Sabin vaccine strain was used (mutant S.3). This point mutation is not supposed to seriously alter RNA secondary structure because a U-G base pair can substitute for the original C-G base pair in the folded IRES (Fig. 5C). With these mutations, we observed again that both mutations affected the translation not only of the downstream Fluc gene but also of the upstream CAT gene (Fig. 5E, lanes 8 and 9) or the upstream Rluc gene (lanes 11 and 12). These results confirm our above conclusions that IRES function is the parameter that enhances upstream reporter gene activity at elevated salt concentrations.

Translation Enhancement in Vivo—To investigate whether this translation enhancement is a biologically relevant effect also under the physiological conditions in living cells, we transfected RNAs transcribed from the dicistronic constructs into cell lines. We used BHK21 cells for FMDV because this virus replicates well in these cells (44), whereas for the poliovirus IRES we used HeLa cells that are known to support poliovirus replication (45–47). Either the RNAs were used unmodified after in vitro transcription (Fig. 5, H and I), or they were capped and polyadenylated before transfection to mimic cellular mRNA modifications (Fig. 5, L and M).

In these experiments, translation enhancement was detected as a higher level of first reporter expression from a dicistronic mRNA containing an active IRES in comparison with a similar mRNA with an inactive IRES. Not surprisingly, the degree of translation enhancement in the presence of a given active IRES correlates with the capacity of the respective IRES to enhance translation in RRL (compare Fig. 2). In particular, translation was 2–3.5-fold higher in the presence of an active FMDV IRES (Fig. 5, H and L), whereas the poliovirus IRES showed only a lower degree of translation enhancement also in living cells (Fig. 5, I and M). This lower degree of translation enhancement with the poliovirus IRES is consistent with our above finding that it is less active in translation enhancement compared with the FMDV IRES also in the in vitro assays (Figs. 2 and 5E). The reason for this lower activity of the poliovirus IRES compared with the FMDV IRES in this assay may be that the poliovirus IRES has its salt optimum at about 80 mM K+, whereas the FMDV IRES has its optimum at about 120 mM (36). Consequently, the poliovirus IRES exhibits a lower degree of translation enhancement than the FMDV IRES under the potassium conditions in living cells of about 140 mM. Moreover, when the dicistronic mRNA is capped and polyadenylated and therefore first reporter activity is high anyway, the weaker poliovirus IRES can additionally contribute to first reporter activity by translation enhancement only marginally (Fig. 5M). Northern blots (Fig. 5, J and K) show that differences in RNA stability do not account for the differences in first reporter expression (ethidium bromide stains were always used to check sample RNA integrity and amounts; example shown on left side of Fig. 5J).


Figure 5
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  		FIGURE 5.
IRES function is required for translation enhancement. A, dicistronic expression cassettes. The arrows indicate IRES mutations. The dotted lines indicate PCR fragments used as transcription templates in G. B, mutations in the stem-loop 4 of the FMDV IRES. C, mutations in the poliovirus IRES. The S.3 mutant has a C472U exchange in the stem-loop V. In the {Delta}V mutant, the entire stem-loop V was deleted. D and E, 35S-labeled proteins from in vitro translation of dicistronic RNAs in RRL. The RNAs contain the wt FMDV IRES and its mutant variants (lanes 1–6) or the PV IRES and its mutant variants (lanes 7–12). D, translation at 50 mM K+. E, translation at 120 mM K+ for FMDV (lanes 1–6) and 80 mM K+ for poliovirus (lanes 7–12). F, Northern blot of RNAs analyzed after re-extraction from translation reactions as in E using an Fluc antisense probe. G, in vitro translations as in E but using RNAs transcribed from PCR fragments that do not contain second reporter gene sequences (dotted lines in A). H, I, L, and M, first reporter Rluc (black bars, R) and second reporter Fluc (gray bars, F) expression measured 4 h after transfection of dicistronic RNAs (shown in A) into cell lines. Rluc expression values were normalized to co-transfected beta-galactosidase mRNA. All of the experiments were performed at least three times. H and I, uncapped and nonpolyadenylated RNAs containing the FMDV IRES or its mutant variants in BHK21 cells (H) or the poliovirus IRES and its variants in HeLa cells (I). Expression with the wt IRES was set to 100%, and expression with an "empty" dicistronic mRNA without IRES is shown (no IRES). J and K, Northern blots for RNAs re-extracted from transfected cells as in H and I, together with an ethidium bromide stain of a re-extracted RNA sample and DNA marker (M; 600, 800, 1000, 1500, 2000, 2500, and 3000 bp). L and M, capped and polyadenylated RNAs containing the FMDV IRES or its mutant variants in BHK21 cells (L) or the poliovirus IRES and its variants in HeLa cells (M).

 


Figure 6
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  		FIGURE 6.
Translation enhancement requires eIF4F binding, not IRES-driven translation initiation activity. A, the dicistronic mRNA transcribed from plasmid pHPI933 carries the CAT gene as first reporter, the HCV IRES, and the Fluc gene as second reporter. The dotted line indicates the {Delta}3abc mutation in the HCV IRES. B, 35S-labeled Fluc and CAT proteins from in vitro translation in RRL of dicistronic mRNA transcribed from pHPI933 at different K+ concentrations. C, CAT protein expressed from a dicistronic RNA as in B but lacking the stem-loops 3a, b, and c (indicated in A). D, dicistronic mRNA with the FMDV IRES as in Fig. 1A but with a precise deletion of stem-loop 3. The asterisk indicates the initiation factor-binding site in stem-loop 4. E, 35S-labeled CAT protein from in vitro translation in RRL of dicistronic mRNA transcribed from pCAT-FM-d3 (see A) at different K+ concentrations. F, CAT protein expressed from pcDNA3.1(+)/CAT RNA (see D).

 
Translation of the Second Reporter Is Not Required for Translation Enhancement—It could be hypothesized that more efficient reutilization of ribosomes terminating near the RNA 3'-end could cause the translation enhancement. Therefore, we analyzed whether translation of the second reporter gene downstream of the IRES is required for the stimulation of upstream reporter translation. For this purpose, we used reporter RNAs in vitro transcribed from PCR fragments that include the first reporter gene and the IRES but no second reporter gene (broken lines in Fig. 5A).

The results show that virtually no translation of the first reporter CAT is possible at 120 mM K+ in the presence of a mutated FMDV IRES (Fig. 5G, lanes 2 and 3), whereas the wild-type FMDV IRES confers efficient upstream gene translation (lane 1). This result is consistent with the previous observation that CAT gene translation is completely abolished at K+ concentrations of more than 110 mM when the gene is present in a monocistronic RNA (Fig. 2D), whereas CAT translation decreases only at K+ concentrations of more than 120 mM K+ in the presence of a downstream FMDV IRES (Fig. 2B). Similar results were obtained when Rluc as first reporter or the poliovirus IRES were used (Fig. 5G), even if overall Rluc expression appeared lower than CAT expression (probably because of the shorter 5'-UTR of the Rluc RNA of 11 nucleotides compared with the longer 5'-UTR of CAT RNA of 51 nucleotides). Taken together, the enhancement of upstream gene translation at least does not require stages of translation initiation subsequent to binding of the small ribosomal subunit to the RNA.

The eIF4F-independent HCV IRES Does Not Confer Translation Enhancement—The above results suggest that either the binding of initiation factors or the mere binding of the small ribosomal subunit confer upstream gene translation enhancement. To distinguish between these two possibilities, we used the HCV IRES in a dicistronic mRNA (Fig. 6A). In contrast to the picornavirus IRES elements, the HCV IRES can bind to the small ribosomal subunit in the absence of virtually any translation initiation factors (19, 20), whereas binding of ribosomes to picornavirus IRES elements requires initiation factors (9, 10, 13, 17, 18). The results of translation at different K+ concentrations (Fig. 6B) show that CAT translation efficiency decreased at 70 mM K+ and was abolished above 80 mM. In contrast, Fluc translation was equally efficient at 50–70 mM K+, decreased only slowly at higher concentrations, and was still detectable at 120 and 130 mM. Thus, the HCV IRES does not confer enhancement of upstream gene translation, indicating that most likely translation initiation factors of the eIF4 group, in particular eIF4F, are involved in the enhancement of upstream gene translation. As a negative control, translation from a dicistronic mRNA with a defective HCV IRES (deletion of stem-loops 3a–c) was analyzed (Fig. 6C).

Translation Enhancement Correlates with the Ability of the IRES to Bind eIF4F, but Not with IRES Activity—To further distinguish between the need for a mere capture of initiation factors or the possible need for subsequent steps of translation initiation for translation enhancement, we used a defective FMDV IRES that lacks the central stem-loop 3 (Fig. 6D). This mutation completely kills IRES activity (5, 48) but leaves the initiation factor-binding site, the stem-loop 4 (asterisk in Fig. 6D), intact (15). With this defective IRES, we clearly observed a strong enhancement of first reporter translation up to 110 or 120 mM K+ (Fig. 6E), whereas CAT expression from a monocistronic mRNA strongly decreased at K+ concentrations above 60 mM (Fig. 6F). Taken together with the above results, this strongly suggests that the stimulation of upstream gene translation by a picornavirus IRES requires the binding of initiation factors (most likely eIF4F) to the IRES but does not necessarily require IRES activity.


Figure 7
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  		FIGURE 7.
eIF4F is the factor involved in translation enhancement. A, structure of the capped and polyadenylated pRL-FM and phRL-null RNAs. B, first reporter Rluc activity measured after in vitro translation in RRL of RNAs shown in A in the absence (black bars) or presence of the eIF4E-inhibitor m7GDP (100 µM) (gray bars). As a control, 100 µM GDP was used (hatched bars). Left panel, Rluc expression from dicistronic pRL-FM RNA. Right panel, Rluc expression from monocistronic phRL-null RNA. C, organization of eIF4G with binding sites for eIF4E, eIF4A, and eIF3 (gray box) and the cleavage site of the FMDV L protease (modified according to Ref. 55). D, Western blot of eIF4G in RRL before (–) or after (+) 45 min incubation with pFMDV14 RNA expressing the L protease, stained with an antiserum against the C-terminal part of eIF4G. E, 35S-labeled proteins from in vitro translation at different K+ concentrations of uncapped dicistronic RNA transcribed from pCAT-FM (see Fig. 1A) in RRL preincubated with L protease RNA.

 
Translation Enhancement Is Mediated by eIF4F—To unambiguously identify eIF4F as the component that acts in the stimulation of upstream gene translation, we used m7GDP, which inhibits binding of the cap-binding eIF4E moiety of initiation factor eIF4F to the 5'-terminal cap nucleotide of mRNAs. We compared the translation efficiencies of Renilla luciferase reporter contained as first reporter either in a dicistronic or in a monocistronic capped and polyadenylated mRNA (Fig. 7A).

At 100 mM K+, the first reporter Rluc was translated with high efficiency when present in a dicistronic mRNA (Fig. 7B, left panel). In contrast, expression of the first reporter was much lower when the gene was present in a monocistronic mRNA (Fig. 7B, right panel), indicating that the downstream IRES enhances first reporter expression. Most importantly, first reporter expression in the dicistronic mRNA was strongly reduced by the addition of m7GDP, which inhibits binding of eIF4E to the cap nucleotide (Fig. 7B, left panel), whereas GDP had no significant effect. This confirms that the cap-binding complex eIF4F is involved in the translation enhancement effect. In contrast, the eIF4F inhibitor hippuristanol completely abolished cap-dependent as well as IRES-dependent translation because it inhibits eIF4F activity by binding to eIF4A (data not shown).

We conclude from this experiment that the tripartite initiation factor complex eIF4F is captured to the dicistronic mRNA by binding to the stem-loop 4 of the FMDV IRES by virtue of its eIF4G moiety (15, 16). After being captured, eIF4F can then be provided in cis to the 5'-end of the mRNA, where its eIF4E moiety can bind to the cap nucleotide or the eIF4G moiety binds to the RNA by its intrinsic RNA-binding domains, and then eIF4F promotes translation initiation of the first reporter gene.

The C-terminal Fragment of eIF4G Can Confer Translation Enhancement on Uncapped mRNA—Our above experiments lead to the conclusion that translation enhancement may act on capped as well as on uncapped mRNAs. Accordingly, it is known not only that eIF4F can bind to mRNA by using its cap-binding eIF4E moiety, but that eIF4F is able to bind to RNA also independently of eIF4E using the central part of its eIF4G moiety (49, 50). Thus, we hypothesized that not only the N-terminal moiety of eIF4G that interacts with the cap-binding protein eIF4E but also the central part of eIF4G could confer translation enhancement.

For this reason, we have treated reticulocyte lysate with the L-protease of FMDV that cleaves off the N-terminal eIF4E-binding moiety of eIF4G (Fig. 7C). Complete eIF4G cleavage was monitored with an antibody directed against the C terminus of eIF4G (Fig. 7D, lane 2). With this lysate containing only cleaved eIF4G, we performed in vitro translation reactions using the uncapped dicistronic mRNA with the FMDV IRES (Fig. 1A) at different K+ concentrations (Fig. 7E). The result shows that first reporter CAT is efficiently expressed at salt concentrations of up to 120 mM and even above, in marked contrast to the decrease of CAT expression from a monocistronic mRNA at salt concentrations above 80 mM (compare Fig. 2D).

We conclude that, whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES by its eIF4G moiety, it can be provided in cis to the RNA 5'-end and there act in translation initiation, either by binding to the cap nucleotide with its eIF4E moiety or by binding to the RNA cap-independently by its eIF4G moiety.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that internal ribosome entry sites can enhance translation not only of the downstream gene but also of a gene upstream of the IRES at salt concentrations that are comparable with those present in living cells. This translation enhancement works with picornavirus IRES elements that utilize the canonical translation initiation factors for their activity (9). In contrast, this translation enhancement does not work with the HCV IRES, which can bind directly to the small ribosomal subunit without binding eIF4 initiation factors (19, 20).

Our results show that the initiation factor eIF4F, in particular its large subunit eIF4G, is involved in the translation enhancement effect. The central part of eIF4G that lacks the eIF4E-binding site can drive translation (49), and cleavage with different viral proteases led to mapping of an RNA-binding region in the central core domain of the eIF4G protein (50) that is separate from the binding site for the cap-binding protein eIF4E. Additionally, the central core domain of eIF4G is capable of binding to the EMCV IRES by protein determinants (51) even different from those described by Prévôt and co-workers (50) and drives initiation complex formation with the EMCV IRES (9). Thus, the eIF4G protein on its own can bind to RNA via two intrinsic RNA-binding determinants in addition to the generalized binding of eIF4G to all capped mRNAs conferred by the associated eIF4E protein, even though binding of eIF4E decreases the affinity of eIF4G for uncapped mRNAs by conformational changes (52). Accordingly, the translation of cellular mRNAs has been reported to also occur cap-independently when the concentrations of general RNA-binding proteins are low, whereas at high concentrations of these proteins the RNA would be inaccessible for the intrinsic RNA-binding domain of eIF4G (53).

In the case of the translation enhancement effect we report here, this intrinsic RNA binding capacity of the eIF4G protein itself appears to be sufficient to confer binding of the protein to the mRNA region in question. Transfer of the eIF4G protein from the picornavirus IRES as an initial "initiation factor capture site" in the RNA to another site at the 5'-terminus of the RNA then occurs with a certain efficiency when the local concentration of the second RNA-binding site is high enough, i.e. when it is present on the same dicistronic mRNA molecule in cis.

This transfer becomes evident only under conditions at which the efficiency of translation of the first reporter gene would be low if placed in a monocistronic mRNA. At low salt concentration, a possible delivery of initiation factors from the IRES to the upstream gene plays a negligible role because the upstream gene is translated efficiently anyway. In contrast, at high salt concentrations the upstream gene is translated inefficiently if placed in a monocistronic mRNA or in a dicistronic mRNA with a mutant downstream IRES. Only when an IRES that is competent to bind eIF4G acts as a high affinity capture site in the dicistronic mRNA, is translation of the upstream first reporter gene efficient because the downstream IRES supplies initiation factors in cis.

The findings presented here may be important preferentially for the design of replicon constructs used for the analysis of translation and replication of positive strand RNA viruses and the interpretation of results obtained with these constructs. As a consequence of the translation enhancement effect, an internal picornavirus IRES in a replicon system or reporter construct may influence the activity of upstream genes in cis and by that may conceal the modulating effects of mutations in the viral sequences on translation efficiency that would not escape detection when the element in question would be investigated in a monocistronic mRNA system.

For example, an additional picornavirus IRES in an HCV replicon may level out differences in translation efficiency and by that prevent the assignment of the effect of a given mutation in the viral RNA to the process of translation, whereas an effect of the same mutation on RNA synthesis is detected well by monitoring genome amplification. Thus, the translation enhancement described here may be a reason why some studies using dicistronic replicon constructs did not monitor an effect of mutations in the sequence of the HCV 3'-UTR on translation but assigned the effects of these mutations solely to replication (29, 30); while using monocistronic reporter RNAs, we and others found that mutations in the 3'-UTR can affect translation (43, 54). Thus, it might be helpful for future studies to investigate the effects of mutations in viral cis-acting signals in monocistronic reporter systems and to more clearly assign the effects of these mutations to the different steps of genome replication by the use of separate read-outs for translation and for minusstrand synthesis.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 535 and GK370. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Submitted in partial fulfillment of the requirements for a Ph.D. at Justus-Liebig-University Giessen. Back

2 To whom correspondence should be addressed. Tel.: 49-641-99-47471; Fax: 49-641-99-47429; E-mail: michael.niepmann{at}biochemie.med.uni-giessen.de.

3 The abbreviations used are: IRES, internal ribosome entry site; EMCV, encephalomyocarditis virus; FMDV, foot-and-mouth disease virus; HCV, hepatitis C virus; eIF, eukaryotic translation initiation factor; CAT, chloramphenicol acetyltransferase; UTR, untranslated region; RRL, rabbit reticulocyte lysate; Fluc, firefly luciferase; Rluc, Renilla luciferase; wt, wild type. Back


   ACKNOWLEDGMENTS
 
We thank P. Mavromara (Athens) for plasmid pHPI933, B. Kastner (Göttingen) and A. Miller (Mons) for cytoplasmic HeLa extracts, and J. Tanaka (Okinawa) and J. Pelletier (Montreal) for hippuristanol.



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