Internal translation initiation in the design of improved expression vectors Monique V. Davies and Randal J. Kaufman Genetics Institute, C a m b r i d g e , Massachusetts, U S A The discovery of a novel, cap-independent mechanism of translation used by picornavirus mRNAs has led to new advances in the engineering of mammalian expression vectors. It is now possible to express several proteins in a coordinate fashion from a single mRNA. Improved expression vectors suitable for virus-mediated transfer and direct DNA transfer are described. Current Opinion in Biotechnology 1992, 3:512-51 7
Introduction Gene transfer into eukaryotic cells can be achieved by virus infection or direct DNA transfer. At present retrovirus-mediated transfer is the method of choice for h u m a n gene therapy because of its high efficiency in a variety of cell types. Direct DNA transfer combined with the ability to select and amplify plasmid DNA integrated in the cell g e n o m e is used extensively to engineer stable cell lines expressing high levels of foreign genes. In both cases, the ability to express two or more genes, allowing the expression of subunits of a multimeric protein or of multiple heterologous proteins would b e most advantageous. Although this can be achieved in a variety of ways (such as cotransfection of several vectors, use of vectors containing multiple transcription units or alternative splicing signals) all these methods have shortcomings. Recent advances in the understanding of the unique w a y in which picornaviruses are translated have allowed n e w strategies to permit the translation of several o p e n reading frames (ORFs) from a single transcript. This review will first summarize current k n o w l e d g e of the mechanism of internal translation initiation and then describe its application to the engineering of i m p r o v e d eukaryotic vectors, such as retroviral vectors and plasmid DNA expression vectors, suitable for the stable expression of foreign genes in mammalian cells.
Translation initiation in eukaryotes Eukaryotic mRNAs, unlike their prokaryotic counterparts, are not polycistronic. They lack the Shine-Dalgarno sequence, but their unique 5'-terminal cap structure is required for efficient translation initiation. The
m7G cap is recognized by the cap-binding c o m p l e x eIF-4F in an ATP-dependent manner. U p o n binding of eIF-4F to the 5' end of the mRNA, local unwinding occurs, allowing the 40S ribosomal subunit to bind. Scanning then proceeds, facilitated by the unwinding activity of initiation factors eIF-4A and eIF-4B, in an ATP-dependent fashion. U p o n recognition of the first AUG c o d o n in an appropriate context, the 60S ribosomal subunit joins and protein synthesis is initiated [1,2]. C a p - d e p e n d e n t initiation is regulated b y primary mRNA structural elements, such as the sequence context around the initiation AUG codon and secondary structural elements. The secondary structure near the 5' end of the mRNA can inhibit initiation, w h e r e a s its presence immediately after an AUG codon can stall rib o s o m e s and allow initiation at an AUG that is contained within a p o o r sequence context for efficient initiation. Translation efficiency is also d e p e n d e n t on the availability and integrity of all the translation initiation factors.
Cap-independent translation of picornavirus RNA J
Unlike most eukaryotic mRNAs, picornaviral mRNAs are not capped. A small, virus-encoded oligopeptide (VPg) is attached to the 5'end of the RNA, and is cleaved off before translation, leaving a p h o s p h o r y lated urydilic acid residue. The 5' untranslated regions (5'UTR) of picornaviral mRNAs are very long, about 650-1200 nucleotides (nO, contain many silent AUGs and have extensive secondary structure. In addition, s o m e picornaviruses inactivate the large subunit of the cap-binding c o m p l e x p220 u p o n infection, resulting in the translational inhibition of cellular mRNAs, although
Abbreviations DHFR--dihydrofolate reductase; EMCV--encephalomyocarditis virus; ES---embryonic stem: IRES---internal ribosomal entry site; LTR--Iong terminal repeat; MTX--methotrexate; nt--nucleotide; ORF--open reading frame; RSV---Roussarcoma virus; UTR--untranslated region.
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Internal translation initiation in the design of improved expression vectors Davies and Kaufman 513 viral protein synthesis is unaffected. All these observations suggest that translation initiation of picomaviral mRNAs occurs in a cap-independent manner. The definitive experiments, showing that an element from the 5' UTR of encephalomyocarditis virus (EMCV) or poliovirus RNA inserted between two cistrons of a dicistronic RNA could direct ribosomes to bind internally and initiate translation of the second cistron independently of the first cistron, were reported in 1988 [3-5]. Deletions within this element abolished translation. The c/s-acting element was termed either 'internal ribosomal entry site' ORES), used herein, or 'ribosome landing pad'. Experiments to define the minimal sequence of the EMCV IRES were performed [6-10] and identified two elements: a complex secondary structure of about 450 nt and an oligopyrimidine tract-AUG element that appears to be required to promote ribosome binding (Fig. 1). Ribosome binding in EMCV occurs at the 3' end of the IRES, very close to the initiation codon AUG-11. Similarily, the minimum sequence required for the poliovirus IRES is composed of a complex secondary structure of about 400 nt, as well as an oligopyrimidine-AUG element [11,12]. Initiation at the poliovirus IRES, however, involves ribosome binding followed by scanning to the initiation AUG 154 nt downstream. In both cases, the distance between the oligopyrimidine tract and AUG is critical [7,12]. Besides these cis-acting elements, internal initiation is also dependent on trans-acting cellular factors. To date, two cellular proteins have been identified: p52 and p57. The former, p52, appears to be involved in the function of the oligopyrimidine tract [13], while p57 may mediate binding of the 40S ribosomal subunit to the IRES [6]. Other members of the picornavirus family, such as foot-and-mouth disease virus [14,15] and h u m a n rhinovirus [16], also use an internal entry mechanism, but their t-RESs have not been studied as extensively as those of EMCV and poliovirus. Recently, the 5' leader of a cellular RNA was s h o w n to mediate internal initiation, raising the possibility that internal ribosome binding may also be used by other cellular mRNAs [17]. A recent and thorough review b y Agol [18] covers the present knowledge related to picornavirus 5'UTRs.
Use of internal initiation in virus-mediated gene transfer Virus-mediated gene transfer is the most efficient method used to introduce foreign DNA into a variety of different cell types. The two commonly used viral systems are based on retroviruses and vaccinia virus. Retrovirus-mediated gene transfer is presently the method of choice for gene therapy, because of its high efficiency for a variety of cell types which are difficult to transfect by other methods. Retroviral vectors have been engineered to express the gene of interest as well as a selectable marker, allowing the isolation
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[6]. of productively infected cells which can then be exp a n d e d for subsequent use. In the past, two general types of vectors have been used. The first utilizes two promoters, usually the long terminal repeat (LTR) for expression of the gene of interest and an internal promoter sequence to express a selectable marker gene product [19]. A shortcoming of this method is the competitive interference between promoters, resulting in the expression of one gene or the other, but not both. A second type of vector utilizes viral splicing signals to generate two separate mRNAs. In this case, a shortcoming is that expression of one gene is at the expense of the other, and that splicing efficiency is unpredictable and highly dependent on the sequence of the insert. The first example of the use of internal initiation in a retroviral vector is illustrated in [20"]. The experiments presented by Adam et al. [20"-] demonstrate the coordinate expression of two ORFs from a single transcript in a stable cell line. Dicistronic retroviral vectors were constructed by inserting 5' UTR sequences from either EMCV RNA (nt 260-837) or poliovirus type 2 (Lansing) RNA (nt 1-732) into derivatives of the Moloney murine leukemia virus-based vector LNL6. These vectors were used to infect a variety of cell lines. Expression of the downstream ORF was 25-100% of that observed w h e n
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Expressionsystems placed in the upstream position and the insertion of picornaviral IRES sequences did not interfere with the production of a high titer of virus. The integrity of the dicistronic message was demonstrated. The data presented in this paper support the internal initiation model in a stable in vivo assay, and illustrate the utili W of coexpressing two ORFs in a retroviral vector. The superiority of the use of internal initiation to coexpress two genes, over the use of two promoters or splicing mechanisms was illustrated very elegantly in [21-]. Using an immunohistochemical assay, Ghattas et al. [21..] demonstrated that most cells infected with Rous sarcoma virus (RSV)-based vectors containing the IRES from EMCV expressed both ORFs, while ceils infected with internal promoter or regulated splicing vectors expressed either one or the other. In addition, the IRES-containing vectors allowed more efficient expression of the downstream gene and produced higher viral titers. Finally, vectors encoding the l a c Z marker and a second bioactive gene, v-src, were shown to be useful in the generation of transgenic cells, identified with a histochemical stain for lacZ. LacZ-positive cells displayed abnormal m o r p h o l o g y attributable to vsrc expression. Koo et al. [22.] also reported the use of the IRES sequence of EMCV from nt 260-827 in a spleen necrosis virus-based vector to coexpress two selectable markers. Northern and Southern blot analysis s h o w e d that the provirus was stably maintained in infected cells which had been selected for drug resistance, and a single dicistronic transcript was detected. Finally, Morgan et al. [23-] explored the feasibility of using multiple IRES-ORF cassettes to generate multicistronic mRNAs. Tricistronic vectors were engineered using EMCV and poliovirus IRES. Although the efficiency of expression in the tricistronic vectors was lower than that of dicistronic vectors, these vectors were capable of generating high titer, stable producer cells that could transduce target cells successfully. The ability to translate multiple proteins independently from a single transcript could be very useful in gene therapy. Vaccinia virus-based vector systems are rapidly becoming an important tool to produce recombinant proteins for biological and biochemical analysis (see Moss, this issue, p p 518-522). A hybrid vector system, utilizing highly efficient prokaryotic transcriptional elements such as the T7 promoter and the T7 RNA polymerase within recombinant vaccinia viruses, has been used to express target genes transiently [24]. This expression system is based on the coexpression of two recombinant vaccinia viruses, one containing the T7 RNA polymerase gene under the control of a vaccinia virus promoter, and the other other containing the target gene preceded by the T7 promoter and followed by T7 termination sequence signals. T7 transcripts in this system represented 10-30% of the total cytoplasmic RNA 24 hours after transfection but were found to be very inefficiently capped, resulting in a discrepancy
between protein synthesis and mRNA levels. The efficiency of translation of these transcripts was improved as much as sevenfold by introducing the EMCV IRES between the T7 promoter and the target gene [25]. The level of expression was further improved b y transferring the cells to an hypertonic medium, which favors the translation of uncapped EMCV RNAs over cellular mRNAs. These experiments demonstrate the general utility of the IRES to improve translation for mRNAs that are not capped, for example the mRNAs expressed in RNA viral vector systems (see Rice, this issue, pp 523-532). An interesting use of internal initiation in the engineering of a functional dicistronic poliovirus was demonstrated by Molla et al. [26..]. Poliovirus RNA encodes a polyprotein that is processed by virus-encoded proteinases. A dicistronic poliovirus was engineered by introducing the EMCV IRES at the junction of the polypeptides P1 and P2, which are normally cleaved by the polio proteinase 2APto. The recombinant poliovirus was infectious and the time course of appearance of viral polypeptides and host protein synthesis shut-off was almost identical to the wild-type poliovirus. The ability, through the use of an IRES structure, to dissect the role of precursors in the processing of a viral polyprotein is novel and could be used to the same effect in assessing the role of various domains of any polypeptide. In addition, the ability to duplicate the IRES function in a single virus will allow a unique approach to the identification of structurally important features within the IRES by mutational analysis.
Use of internal initiation in DNA-mediated gene transfer DNA-mediated gene transfer is frequently used to obtain high level expression of heterologous genes in mammalian ceils [27]. Cotransfection of a selectable marker gene with the gene of interest allows the selection of ceils that express both the gene of interest and the selectable marker. This method, called cotransformarion [28], yields variable frequencies of coexpression depending On the cell line and the method of transfection. Vectors that contain two separate transcription units, or a single transcription unit in which the selectable marker is positioned within the 3" end of the mRNA, have proven to be useful for the expression of foreign genes [29,30]. In the case of dicistronic mRNA expression vectors, translation of the selectable marker is very inefficient and is thought to occur through a translational termination-reinitiation mechanism. These vectors have b e e n used successfully to derive highly amplified cell lines as high levels of amplification are necessary for the cells to survive selection. The drawback is that the frequency of transformation is low, due to the inability of cells to survive selection, and ceils often delete or rearrange the 5' cistron w h e n selective pressure is applied.
Internal translation initiation in the design of improved expression vectors Davies and Kaufman The limitations o f dicistronic v e c t o r s have b e e n o v e r c o m e b y the u s e o f the EMCV IRES to p r o m o t e internal initiation o f the s e c o n d cistron [30,31"']. A n e w set o f e x p r e s s i o n vectors h a s b e e n g e n e r a t e d to p r o m o t e efficient internal translation o f selectable m a r k ers, s u c h as d i h y d r o f o l a t e r e d u c t a s e (DHFR) pED, a n d a m e t h o t r e x a t e - r e s i s t a n t DHFR ( p E D - m t x r) [31"'], as w e l l as a d e n o s i n e d e a m i n a s e (pEA) a n d n e o m y c i n p h o s p h o t r a n s f e r a s e ( p E D 4 - N e o ) [30]. These v e c t o r s are r e p r e s e n t e d in Fig. 2. T h e pED a n d p E D - m t x r v e c tors w e r e u s e d successfully to d e r i v e highly a m p l i f i e d cell lines [31"]. G r e a t e r t r a n s f o r m a t i o n efficiencies, e a s e of s e l e c t i o n d u e to the ability to p o o l large n u m b e r o f transformants, a n d the u s e o f s e q u e n t i a l i n c r e a s e s in m e t h o t r e x a t e (MTX) to select for a p o p u l a t i o n o f ceils that h a s a m p l i f i e d the g e n e , m a k e t h e s e vectors u s e f u l for a variety o f p u r p o s e s . T h e y are e q u a l l y efficient in transient a n d in stable transfection, a n d the f r e q u e n c y o f d e l e t i o n s o r r e a r r a n g m e n t s is m u c h l o w e r t h a n w i t h dicistronic v e c t o r s that d o n o t c o n t a i n the EMCV IRES. The p E D - m t x r v e c t o r can b e u s e d in a variety o f cells as the DHFR in it c o n t a i n s a l e u c i n e to arginine m u t a t i o n at r e s i d u e 22, a n d e n c o d e s a n e n z y m e that is s e v e r a l h u n d r e d - f o l d m o r e resistant to MTX. Ceils that are n o t deficient in D H F R are initially s e l e c t e d with a h i g h e r a m o u n t o f MTX (usually 0.3 bLM), so that only t h o s e that w e r e successfully t r a n s f e c t e d survive.
m a r k e r for the p E D v e c t o r s w e r e a n a l y z e d [32"]. T h e A U G c o d o n of DHFR w a s either f u s e d to the AUG-11 or AUG-12 of EMCV, or p l a c e d in frame with AUG-11 w i t h a X h o I linker. T h e m a j o r site o f initiation o c c u r r e d at AUG-11, but initiation at d o w n s t r e a m AUGs c o u l d also b e detected. T h e s e q u e n c e c o n t e x t of AUG-11 i n f l u e n c e s the initiation at AUG-11 as well as that o f d o w n s t r e a m AUGs, a n d a p u r i n e at p o s i t i o n - 3 is r e q u i r e d as in c a p - d e p e n d e n t initiation. T h e s e studies s h o w that, w h e n d e s i g n i n g polycistronic vectors, t h o u g h t must b e g i v e n to the d e s i g n of the j u n c t i o n b e t w e e n the IRES a n d the g e n e f o l l o w i n g it in o r d e r to m a x i m i z e e x p r e s s i o n o f that g e n e a n d m i n i m i z e d o w n s t r e a m initiation. T h e EMCV IRES w a s also u s e d in a dicistronic c o n s t r u c t c o n s i s t i n g of a p r o m o t e > d r i v e n n e o g e n e f o l l o w e d b y a n EMCV I R E S - l a c Z cassette to e x p r e s s n e o a n d l a c Z g e n e s stably in e m b r y o n i c stem (ES) ceils [33"]. S u b s e q u e n t l y , ES cells w e r e m i c r o i n j e c t e d into m o u s e blastocysts, a n d c h i m e r i c e m b r y o s w e r e d e r i v e d b y t r a n s p l a n t a t i o n into p s e u d o p r e g n a n t female mice. B o t h g e n e s w e r e s h o w n to b e translated into functional p r o teins in ES cells, a n d the chimeric e m b r y o s also w e r e s h o w n to e x p r e s s ~ - g a l a c t o s i d a s e . A l t h o u g h g e r m - l i n e t r a n s m i s s i o n w a s n o t o b s e r v e d in this e x p e r i m e n t , this a p p r o a c h c o u l d b e a p p l i e d to p r o d u c e transgenic m i c e in w h i c h two g e n e s are e x p r e s s e d coordinately.
T h e effect o f t h e s e q u e n c e c o n t e x t a n d use o f t h e EMCV initiation c o d o n relative to that of the s e l e c t a b l e
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Expressionsystems sequence-replacement vector, containing a human T1 constant-region sequence upstream of an EMCV IRES-neo cassette flanked b y genomic /.t sequences, is used to target the h u m a n Cy 1 constant-region sequence to the immunoglobulin heavy-chain locus in hybridoma cells. The resulting cell lines express a chimeric murine VDJH-human Cy I heavy chain at a level equal to or greater than that of the parental cell line and that level was maintained o v e r a 6-month period. This approach should prove to b e of general use in homologous recombination experiments to alter specific DNA sequences in m a m m a l i a n cells.
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Conclusion The advances in the field of picornavims translation have not only brought rich rewards in the understanding of viral reproduction, but h a v e also increased our knowledge of the eukaryotic translational apparatus. We can expect that more e x a m p l e s of internal initiation will be found in cellular mRNAs. O n c o g e n e s are possible candidates as their 5' UTRs are very long and contain m a n y AUG triplets. As the knowledge of picornavirus translation has o p e n e d n e w avenues in the engineering of m a m m a l i a n expression vectors, the study of IRES-directed translation in stable cell lines might shed light on the factors required for efficient initiation at the IRES. For example, h o w does the modification of initiation factors, brought about by serum deprivation, heat shock or mitosis, affect internal initiation c o m p a r e d to c a p - d e p e n d e n t translation? A better understanding of the interaction between cis-elements of the IRES and protein trans-factors is n e e d e d to clarify the process of internal ribosomebinding in eukaryotic mRNAs further.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1. 2.
KOZAKM: T h e S c a n n i n g M o d e l f o r Translation: A n Update. J Cell Biol 1989, 108:229-241. DONAHUETF: S c a n n i n g , I n t e r n a l Initiation a n d t h e Control o f t h e I n i t i a t i o n o f P r o t e i n Synthesis. Curr Opin Cell Biol 1990, 2:1087-1091.
3.
JANG SK, KRAUSSLICH HG, NICKLIN MJH, DUKE GM, PALMENBERG AC, WIMMER E: A S e g m e n t o f t h e 5' N o n t r a n s l a t e d R e g i o n o f E n c e p h a l o m y o c a r d i t i s Virus RNA Directs I n t e r n a l E n t r y o f R i b o s o m e s D u r i n g I n Vitro Translation. J Virol 1988, 62:2636-2643.
4.
JANG SK, DAVIESMV, I(AUFMANRJ, WIMMERE: I n i t i a t i o n of P r o t e i n S y n t h e s i s b y i n t e r n a l E n t r y o f R i b o s o m e s into t h e 5" N o n t r a n s l a t e d R e g i o n o f E n c e p h a l o m y o c a r d i t i s V i r u s R N A I n Vivo. J Virol 1989, 63:1651-1660.
20. •.
ADAMMA, RAMESHN, MILLERAD, OSBORNE WRA: I n t e r n a l Initiation o f T r a n s l a t i o n i n Retroviral Vectors Carryi n g P i c o r n a v i r u s 5' N o n t r a n s l a t e d R e g i o n s . J Viro11991, 65:4985-4990. This article demonstrates that retroviral vectors containing a poliovirus IRES can express two genes from a single transcript in a stable cell line, and that the 5' UTR poliovirus sequence does not interfere with the production of high virus titer. 21. •.
GHATrAS IR, SANES JR, MAJORS JE: T h e E n e e p h a l o m y o carditis Virus I n t e r n a l RtlJosome E n t r y Site Allows Efficient Coexpression of Two Genes from a Recombi-
Internal translation initiation in the design o f improved expression v e c t o r s Davies and Kaufman 5 1 7 nant P r o v i r u s i n C u l t u r e d Cells a n d in E m b r y o s . Mol
30.
Cell Biol 1991, 11:5848-5859. The use of the EMCV IRES in an RSV-derived vector is s h o w n to provide the most efficient means to coexpress two genes from a single provirus. Vectors encoding the lacZ gene and an IRES-v-src cassette were used in the generation of transgenic cells which can be identified with a histochemical stain and studied for their phenotype due to v-src expression.
KAUFMANRJ: T r a n s l a t i o n a l Efficiency o f P o l y c i s t r o n i c mRNAs a n d t h e i r Utilization to Express Heterologous Genes i n M a m m a l i a n Cells. EMBO J 1987, 6:187-193.
31. •.
KAUFMANRJ, DAVIES MV, WASLEY LC, MICHNICK D: Iln-
22.
K o o HM, BROWN AMC, KAUFMANRJ, PROROCK CM, RON Y, DOUGHERTYJP: A Spleen Necrosis V i r u s - b a s e d R e t r o v i r a l Vector w h i c h E x p r e s s e s T w o G e n e s f r o m a D i c i s t r o n i c MRNA. Virology 1992, 186:669-675. Another example of the use of the EMCV IRES in a retroviral vector. 23. •.
MORGANRA, COUTURE L, ELROY-STEIN O, RAGHEB J, Moss B, ANDERSON "WE: R e t r o v i r a l Vectors C o n t a i n i n g Putative Internal Ribosome Entry Sites: D e v e l o p m e n t o f a P o l y e i s t r o n i c G e n e T r a n s f e r System and AppHcat i o u s to H u m n n G e n e T h e r a p y . Nucl Acids Res 1992, 20:1293-1299. First example of the use of multiple IRES in retroviral vectors. Stable producer cell lines were established with tricistronic vectors, and were s h o w n to synthesize all three gene products and to produce high virus titer that could productively transduce 3T3 cells. 24.
FUERSTTR, NILES EG, STUDIER FW, MOSS B: E u k a r y o t i e
Transient-expression System Based o n R e c o m b i n a n t Vaccinia V i r u s t h a t S y n t h e s i z e s B a c t e r i o p h a g e T7 RNA P o l y m e r a s e . Proc Natl Acad Sci USA 1986, 83:8122~8126. 25.
ELROY-STEINO, FUERST TR, MOSS B: Cap-Independent T r a n s l a t i o n o f MILNA C o n f e r r e d b y E n c e p h a l o m y o carditis V i r u s 5' S e q u e n c e I x n p r o v e s t h e Performance o f t h e Vaceinia V i r u s / B a c t e r i o p h a g e T7 H y b r i d Expression S y s t e m . Proc Natl Acad Sci USA 1989, 86:612645130.
26. .,
MOLLAA, JANG SK, PAUL AV, REUER Q, WIMMERE: Cardiovir a l I n t e r n a l R i b o s o m a l E n t r y Site is F u n c t i o n a l i n a Genetically E n g i n e e r e d D i c i s t r o n i e Poliovirus. Nature 1992, 356:255-257. Insertion of the EMCV IRES into the ORF of the poliovirus polyprorein created a functional dicistronic poliovirus. This w o r k provides unambiguous evidence that the EMCV IRES can initiate translation internally in vivo. It also provides a strategy to analyze the processing of viral polyproteins or the rote of various domains in a polypeptide. 27.
KAUFMAN RJ:
Expression
Strategies for in Mnmmnllan
O b t a i n i n g H i g h Level Cells. Technique 1990,
2:221--236. 28.
WIGLERM. SWEET R, SIM GK, WOLD B, PELLICER A, LACY E, MANIATIST, SILVERSTEINS, AXEL R: T r a n s f o r t n a t i o n o f Mammallaq Ceils w i t h G e n e s f r o m P r o M o t e s and E u k a r y o t e s . Cell 1979, 16:777-785.
29.
KAUFMANRJ: Selection and C o a m p l i f i c a t i o n o f Heterologous Genes i n M a m m a l i a n Cells. Methods in Enzymol 1990, 185:537-566.
proved Vectors for Stable Expression o f Foreign Genes i n M a m m a l i a n Cells b y u s e o f t h e Untranslated
Leader S e q u e n c e f r o m EMC Virus. Nucl Acids Res 1991, 16:4485~i490. A family of dicistronic expression vectors containing the EMCV IRES is described. Efficient expression of the gene of interest is obtained from the adenovirus major late promoter and the expression of the selectable markers (DHFR and DHFR-mtxr), positioned downstream of the EMCV IRES, is independent from the upstream ORF. These vectors are suitable for transient or stable expression of foreign genes in mammalian cells. They yield high transformation efficiencies, are readily amplifiable and are less likely to rearrange or delete the gene of interest. 32.
DAVIESMD, KAUFMAN RJ: T h e S e q u e n c e C o n t e x t o f t h e I n i t i a t i o n C o d o n i n t h e E n e e p h a l o m y o c a r d i t i s Virus Leader Modulates Efficiency o f I n t e r n a l T r a n s l a t i o n Initiation. J Virol 1992, 66:1924-1932. This article studies the effect of the sequence context on translation initiation promoted by the EMCV IRES. Initiation was s h o w n to occur mainly at EMCV AUG-11 but is leaky. The sequence context downstream of EMCV AUG-11 influences the efficiency of initiation at that site as well as that of downstream AUGs. A purine at position - 3 is desirable, as in cap-dependent initiation. KIM DG, KANG HM, JANG SK, SHIN MS: Construction o f a B i f u n c t i o n a l mRNA i n t h e M o u s e b y Using t h e Intern a l R i b o s o m a l E n t r y Site o f t h e E n c e p h a l o m y o c a r d i t i s Virus. Mol Cell Biol 1992, 12:3636-3643. A dicistronic mRNA is s h o w n to be functional in mouse ES cells as well as in the chimeric embryos derived from them. This approach could be used to produce transgenic mice in which two genes can be expressed coordinately. 33.
34. •.
W O O D CR, MORmS GE, ALDERMAN EM, FOUSER L, KAUFMAN RJ: A n I n t e r n a l R i b o s o m e B i n d i n g Site can b e Used to Select for H o m o l o g o u s Recombinants at art Inlm u n o g l o b n l i n H e a v y - c h a i n Locus. Proc Natl Acad Sci USA 1991, 88:8006-8010. A sequence replacement-type vector containing a h u m a n T1 constant region sequence followed by an EMCV IRES-neo cassette is described. Flanking sequences homologous to murine genomic regions target that vector to the immunoglobulin heavy-chain locus. The strategy of using an EMCV IRES-neo cassette has potential application to a range of gene targeting vectors.
MV Davies and RJ Kaufman, Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts, MA 02140, USA.
Introduction Gene transfer into eukaryotic cells can be achieved by virus infection or direct DNA transfer. At present retrovirus-mediated transfer is the method of choice for h u m a n gene therapy because of its high efficiency in a variety of cell types. Direct DNA transfer combined with the ability to select and amplify plasmid DNA integrated in the cell g e n o m e is used extensively to engineer stable cell lines expressing high levels of foreign genes. In both cases, the ability to express two or more genes, allowing the expression of subunits of a multimeric protein or of multiple heterologous proteins would b e most advantageous. Although this can be achieved in a variety of ways (such as cotransfection of several vectors, use of vectors containing multiple transcription units or alternative splicing signals) all these methods have shortcomings. Recent advances in the understanding of the unique w a y in which picornaviruses are translated have allowed n e w strategies to permit the translation of several o p e n reading frames (ORFs) from a single transcript. This review will first summarize current k n o w l e d g e of the mechanism of internal translation initiation and then describe its application to the engineering of i m p r o v e d eukaryotic vectors, such as retroviral vectors and plasmid DNA expression vectors, suitable for the stable expression of foreign genes in mammalian cells.
Translation initiation in eukaryotes Eukaryotic mRNAs, unlike their prokaryotic counterparts, are not polycistronic. They lack the Shine-Dalgarno sequence, but their unique 5'-terminal cap structure is required for efficient translation initiation. The
m7G cap is recognized by the cap-binding c o m p l e x eIF-4F in an ATP-dependent manner. U p o n binding of eIF-4F to the 5' end of the mRNA, local unwinding occurs, allowing the 40S ribosomal subunit to bind. Scanning then proceeds, facilitated by the unwinding activity of initiation factors eIF-4A and eIF-4B, in an ATP-dependent fashion. U p o n recognition of the first AUG c o d o n in an appropriate context, the 60S ribosomal subunit joins and protein synthesis is initiated [1,2]. C a p - d e p e n d e n t initiation is regulated b y primary mRNA structural elements, such as the sequence context around the initiation AUG codon and secondary structural elements. The secondary structure near the 5' end of the mRNA can inhibit initiation, w h e r e a s its presence immediately after an AUG codon can stall rib o s o m e s and allow initiation at an AUG that is contained within a p o o r sequence context for efficient initiation. Translation efficiency is also d e p e n d e n t on the availability and integrity of all the translation initiation factors.
Cap-independent translation of picornavirus RNA J
Unlike most eukaryotic mRNAs, picornaviral mRNAs are not capped. A small, virus-encoded oligopeptide (VPg) is attached to the 5'end of the RNA, and is cleaved off before translation, leaving a p h o s p h o r y lated urydilic acid residue. The 5' untranslated regions (5'UTR) of picornaviral mRNAs are very long, about 650-1200 nucleotides (nO, contain many silent AUGs and have extensive secondary structure. In addition, s o m e picornaviruses inactivate the large subunit of the cap-binding c o m p l e x p220 u p o n infection, resulting in the translational inhibition of cellular mRNAs, although
Abbreviations DHFR--dihydrofolate reductase; EMCV--encephalomyocarditis virus; ES---embryonic stem: IRES---internal ribosomal entry site; LTR--Iong terminal repeat; MTX--methotrexate; nt--nucleotide; ORF--open reading frame; RSV---Roussarcoma virus; UTR--untranslated region.
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© Current Biology Ltd ISSN 0958-1669
Internal translation initiation in the design of improved expression vectors Davies and Kaufman 513 viral protein synthesis is unaffected. All these observations suggest that translation initiation of picomaviral mRNAs occurs in a cap-independent manner. The definitive experiments, showing that an element from the 5' UTR of encephalomyocarditis virus (EMCV) or poliovirus RNA inserted between two cistrons of a dicistronic RNA could direct ribosomes to bind internally and initiate translation of the second cistron independently of the first cistron, were reported in 1988 [3-5]. Deletions within this element abolished translation. The c/s-acting element was termed either 'internal ribosomal entry site' ORES), used herein, or 'ribosome landing pad'. Experiments to define the minimal sequence of the EMCV IRES were performed [6-10] and identified two elements: a complex secondary structure of about 450 nt and an oligopyrimidine tract-AUG element that appears to be required to promote ribosome binding (Fig. 1). Ribosome binding in EMCV occurs at the 3' end of the IRES, very close to the initiation codon AUG-11. Similarily, the minimum sequence required for the poliovirus IRES is composed of a complex secondary structure of about 400 nt, as well as an oligopyrimidine-AUG element [11,12]. Initiation at the poliovirus IRES, however, involves ribosome binding followed by scanning to the initiation AUG 154 nt downstream. In both cases, the distance between the oligopyrimidine tract and AUG is critical [7,12]. Besides these cis-acting elements, internal initiation is also dependent on trans-acting cellular factors. To date, two cellular proteins have been identified: p52 and p57. The former, p52, appears to be involved in the function of the oligopyrimidine tract [13], while p57 may mediate binding of the 40S ribosomal subunit to the IRES [6]. Other members of the picornavirus family, such as foot-and-mouth disease virus [14,15] and h u m a n rhinovirus [16], also use an internal entry mechanism, but their t-RESs have not been studied as extensively as those of EMCV and poliovirus. Recently, the 5' leader of a cellular RNA was s h o w n to mediate internal initiation, raising the possibility that internal ribosome binding may also be used by other cellular mRNAs [17]. A recent and thorough review b y Agol [18] covers the present knowledge related to picornavirus 5'UTRs.
Use of internal initiation in virus-mediated gene transfer Virus-mediated gene transfer is the most efficient method used to introduce foreign DNA into a variety of different cell types. The two commonly used viral systems are based on retroviruses and vaccinia virus. Retrovirus-mediated gene transfer is presently the method of choice for gene therapy, because of its high efficiency for a variety of cell types which are difficult to transfect by other methods. Retroviral vectors have been engineered to express the gene of interest as well as a selectable marker, allowing the isolation
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Fig. 1. Secondary structure of the encephalomyocarditis virus 5' untranslated region from the poly (C) stretch to the AUG i n i t i a t i o n codon of the encephalomyocarditis virus polyprotein shown in a box. Putative binding sites for polypeptides p57 and X, as w e l l as for the 40S ribosomal subunit are shown. From Jang and Wimmer
[6]. of productively infected cells which can then be exp a n d e d for subsequent use. In the past, two general types of vectors have been used. The first utilizes two promoters, usually the long terminal repeat (LTR) for expression of the gene of interest and an internal promoter sequence to express a selectable marker gene product [19]. A shortcoming of this method is the competitive interference between promoters, resulting in the expression of one gene or the other, but not both. A second type of vector utilizes viral splicing signals to generate two separate mRNAs. In this case, a shortcoming is that expression of one gene is at the expense of the other, and that splicing efficiency is unpredictable and highly dependent on the sequence of the insert. The first example of the use of internal initiation in a retroviral vector is illustrated in [20"]. The experiments presented by Adam et al. [20"-] demonstrate the coordinate expression of two ORFs from a single transcript in a stable cell line. Dicistronic retroviral vectors were constructed by inserting 5' UTR sequences from either EMCV RNA (nt 260-837) or poliovirus type 2 (Lansing) RNA (nt 1-732) into derivatives of the Moloney murine leukemia virus-based vector LNL6. These vectors were used to infect a variety of cell lines. Expression of the downstream ORF was 25-100% of that observed w h e n
514
Expressionsystems placed in the upstream position and the insertion of picornaviral IRES sequences did not interfere with the production of a high titer of virus. The integrity of the dicistronic message was demonstrated. The data presented in this paper support the internal initiation model in a stable in vivo assay, and illustrate the utili W of coexpressing two ORFs in a retroviral vector. The superiority of the use of internal initiation to coexpress two genes, over the use of two promoters or splicing mechanisms was illustrated very elegantly in [21-]. Using an immunohistochemical assay, Ghattas et al. [21..] demonstrated that most cells infected with Rous sarcoma virus (RSV)-based vectors containing the IRES from EMCV expressed both ORFs, while ceils infected with internal promoter or regulated splicing vectors expressed either one or the other. In addition, the IRES-containing vectors allowed more efficient expression of the downstream gene and produced higher viral titers. Finally, vectors encoding the l a c Z marker and a second bioactive gene, v-src, were shown to be useful in the generation of transgenic cells, identified with a histochemical stain for lacZ. LacZ-positive cells displayed abnormal m o r p h o l o g y attributable to vsrc expression. Koo et al. [22.] also reported the use of the IRES sequence of EMCV from nt 260-827 in a spleen necrosis virus-based vector to coexpress two selectable markers. Northern and Southern blot analysis s h o w e d that the provirus was stably maintained in infected cells which had been selected for drug resistance, and a single dicistronic transcript was detected. Finally, Morgan et al. [23-] explored the feasibility of using multiple IRES-ORF cassettes to generate multicistronic mRNAs. Tricistronic vectors were engineered using EMCV and poliovirus IRES. Although the efficiency of expression in the tricistronic vectors was lower than that of dicistronic vectors, these vectors were capable of generating high titer, stable producer cells that could transduce target cells successfully. The ability to translate multiple proteins independently from a single transcript could be very useful in gene therapy. Vaccinia virus-based vector systems are rapidly becoming an important tool to produce recombinant proteins for biological and biochemical analysis (see Moss, this issue, p p 518-522). A hybrid vector system, utilizing highly efficient prokaryotic transcriptional elements such as the T7 promoter and the T7 RNA polymerase within recombinant vaccinia viruses, has been used to express target genes transiently [24]. This expression system is based on the coexpression of two recombinant vaccinia viruses, one containing the T7 RNA polymerase gene under the control of a vaccinia virus promoter, and the other other containing the target gene preceded by the T7 promoter and followed by T7 termination sequence signals. T7 transcripts in this system represented 10-30% of the total cytoplasmic RNA 24 hours after transfection but were found to be very inefficiently capped, resulting in a discrepancy
between protein synthesis and mRNA levels. The efficiency of translation of these transcripts was improved as much as sevenfold by introducing the EMCV IRES between the T7 promoter and the target gene [25]. The level of expression was further improved b y transferring the cells to an hypertonic medium, which favors the translation of uncapped EMCV RNAs over cellular mRNAs. These experiments demonstrate the general utility of the IRES to improve translation for mRNAs that are not capped, for example the mRNAs expressed in RNA viral vector systems (see Rice, this issue, pp 523-532). An interesting use of internal initiation in the engineering of a functional dicistronic poliovirus was demonstrated by Molla et al. [26..]. Poliovirus RNA encodes a polyprotein that is processed by virus-encoded proteinases. A dicistronic poliovirus was engineered by introducing the EMCV IRES at the junction of the polypeptides P1 and P2, which are normally cleaved by the polio proteinase 2APto. The recombinant poliovirus was infectious and the time course of appearance of viral polypeptides and host protein synthesis shut-off was almost identical to the wild-type poliovirus. The ability, through the use of an IRES structure, to dissect the role of precursors in the processing of a viral polyprotein is novel and could be used to the same effect in assessing the role of various domains of any polypeptide. In addition, the ability to duplicate the IRES function in a single virus will allow a unique approach to the identification of structurally important features within the IRES by mutational analysis.
Use of internal initiation in DNA-mediated gene transfer DNA-mediated gene transfer is frequently used to obtain high level expression of heterologous genes in mammalian ceils [27]. Cotransfection of a selectable marker gene with the gene of interest allows the selection of ceils that express both the gene of interest and the selectable marker. This method, called cotransformarion [28], yields variable frequencies of coexpression depending On the cell line and the method of transfection. Vectors that contain two separate transcription units, or a single transcription unit in which the selectable marker is positioned within the 3" end of the mRNA, have proven to be useful for the expression of foreign genes [29,30]. In the case of dicistronic mRNA expression vectors, translation of the selectable marker is very inefficient and is thought to occur through a translational termination-reinitiation mechanism. These vectors have b e e n used successfully to derive highly amplified cell lines as high levels of amplification are necessary for the cells to survive selection. The drawback is that the frequency of transformation is low, due to the inability of cells to survive selection, and ceils often delete or rearrange the 5' cistron w h e n selective pressure is applied.
Internal translation initiation in the design of improved expression vectors Davies and Kaufman The limitations o f dicistronic v e c t o r s have b e e n o v e r c o m e b y the u s e o f the EMCV IRES to p r o m o t e internal initiation o f the s e c o n d cistron [30,31"']. A n e w set o f e x p r e s s i o n vectors h a s b e e n g e n e r a t e d to p r o m o t e efficient internal translation o f selectable m a r k ers, s u c h as d i h y d r o f o l a t e r e d u c t a s e (DHFR) pED, a n d a m e t h o t r e x a t e - r e s i s t a n t DHFR ( p E D - m t x r) [31"'], as w e l l as a d e n o s i n e d e a m i n a s e (pEA) a n d n e o m y c i n p h o s p h o t r a n s f e r a s e ( p E D 4 - N e o ) [30]. These v e c t o r s are r e p r e s e n t e d in Fig. 2. T h e pED a n d p E D - m t x r v e c tors w e r e u s e d successfully to d e r i v e highly a m p l i f i e d cell lines [31"]. G r e a t e r t r a n s f o r m a t i o n efficiencies, e a s e of s e l e c t i o n d u e to the ability to p o o l large n u m b e r o f transformants, a n d the u s e o f s e q u e n t i a l i n c r e a s e s in m e t h o t r e x a t e (MTX) to select for a p o p u l a t i o n o f ceils that h a s a m p l i f i e d the g e n e , m a k e t h e s e vectors u s e f u l for a variety o f p u r p o s e s . T h e y are e q u a l l y efficient in transient a n d in stable transfection, a n d the f r e q u e n c y o f d e l e t i o n s o r r e a r r a n g m e n t s is m u c h l o w e r t h a n w i t h dicistronic v e c t o r s that d o n o t c o n t a i n the EMCV IRES. The p E D - m t x r v e c t o r can b e u s e d in a variety o f cells as the DHFR in it c o n t a i n s a l e u c i n e to arginine m u t a t i o n at r e s i d u e 22, a n d e n c o d e s a n e n z y m e that is s e v e r a l h u n d r e d - f o l d m o r e resistant to MTX. Ceils that are n o t deficient in D H F R are initially s e l e c t e d with a h i g h e r a m o u n t o f MTX (usually 0.3 bLM), so that only t h o s e that w e r e successfully t r a n s f e c t e d survive.
m a r k e r for the p E D v e c t o r s w e r e a n a l y z e d [32"]. T h e A U G c o d o n of DHFR w a s either f u s e d to the AUG-11 or AUG-12 of EMCV, or p l a c e d in frame with AUG-11 w i t h a X h o I linker. T h e m a j o r site o f initiation o c c u r r e d at AUG-11, but initiation at d o w n s t r e a m AUGs c o u l d also b e detected. T h e s e q u e n c e c o n t e x t of AUG-11 i n f l u e n c e s the initiation at AUG-11 as well as that o f d o w n s t r e a m AUGs, a n d a p u r i n e at p o s i t i o n - 3 is r e q u i r e d as in c a p - d e p e n d e n t initiation. T h e s e studies s h o w that, w h e n d e s i g n i n g polycistronic vectors, t h o u g h t must b e g i v e n to the d e s i g n of the j u n c t i o n b e t w e e n the IRES a n d the g e n e f o l l o w i n g it in o r d e r to m a x i m i z e e x p r e s s i o n o f that g e n e a n d m i n i m i z e d o w n s t r e a m initiation. T h e EMCV IRES w a s also u s e d in a dicistronic c o n s t r u c t c o n s i s t i n g of a p r o m o t e > d r i v e n n e o g e n e f o l l o w e d b y a n EMCV I R E S - l a c Z cassette to e x p r e s s n e o a n d l a c Z g e n e s stably in e m b r y o n i c stem (ES) ceils [33"]. S u b s e q u e n t l y , ES cells w e r e m i c r o i n j e c t e d into m o u s e blastocysts, a n d c h i m e r i c e m b r y o s w e r e d e r i v e d b y t r a n s p l a n t a t i o n into p s e u d o p r e g n a n t female mice. B o t h g e n e s w e r e s h o w n to b e translated into functional p r o teins in ES cells, a n d the chimeric e m b r y o s also w e r e s h o w n to e x p r e s s ~ - g a l a c t o s i d a s e . A l t h o u g h g e r m - l i n e t r a n s m i s s i o n w a s n o t o b s e r v e d in this e x p e r i m e n t , this a p p r o a c h c o u l d b e a p p l i e d to p r o d u c e transgenic m i c e in w h i c h two g e n e s are e x p r e s s e d coordinately.
T h e effect o f t h e s e q u e n c e c o n t e x t a n d use o f t h e EMCV initiation c o d o n relative to that of the s e l e c t a b l e
A n o t h e r e x a m p l e o f the u s e of an internal r i b o s o m e b i n d i n g site is illustrated b y W o o d et al. [34"]. A
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Fig. 2. Structure of dicistronic mRNA expression vectors containing the encephalomyocarditis virus leader (EMC-L). The selection markers for wildtype dihydrofolate reductase (DHFR), a methotrexate-resistant DHFR, adenosine deaminase (ADA) and neomycin phosphotransferase (Neo) are shown. SV40, simian virus 40; ori, origin of replication; 13-Lac, ~-Iactamase; MLP, major late protein; TPL, tripartitie leader; IVS, intervening sequence.
515
516
Expressionsystems sequence-replacement vector, containing a human T1 constant-region sequence upstream of an EMCV IRES-neo cassette flanked b y genomic /.t sequences, is used to target the h u m a n Cy 1 constant-region sequence to the immunoglobulin heavy-chain locus in hybridoma cells. The resulting cell lines express a chimeric murine VDJH-human Cy I heavy chain at a level equal to or greater than that of the parental cell line and that level was maintained o v e r a 6-month period. This approach should prove to b e of general use in homologous recombination experiments to alter specific DNA sequences in m a m m a l i a n cells.
5.
PELLET1ERJ, SONENBERG N: I n t e r n a l Initiation o f Translation o f Eukaryotic mRNA Directed b y a S e q u e n c e D e r i v e d f r o m Pollovirus RNA. Nature 1988, 334:320-325.
6,
JANG SK, XXTIMMERE: C a p - i n d e p e n d e n t T r a n s l a t i o n o f Enc e p h a l o m y o c a r d i t i s Virus RNA: Structural E l e m e n t s o f I n t e r n a l R i b o s o m a l E n t r y Site a n d I n v o l v e m e n t o f a Cellular 58-kDa RNA-binding Protein. Genes Dev 1990, 4:1560-1572.
7.
JANG SK, PESTOVATV, I~IELLENCUT, WITHERELLGW, WIMMER E: C a p - I n d e p e n d e n t T r a n s l a t i o n o f P i c o r n a v i r a l mRNAs: S t r u c t u r e a n d ~ n c t i o n o f t h e I n t e r n a l R i b o s o m a l E n t r y Site. E n z y m e 1990, 44:292-309.
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KAMINSKIA, HOWELL NIT, JACKSON RJ: Initiation o f Enc e p h a l o m y o c a r d i t i s Virus RNA Translation: t h e Aut h e n t i c I n i t i a t i o n Site is n o t S e l e c t e d b y a S c a n n i n g M e c h a n i s m . EMBO J 1990, 9:3753-3759.
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EVSTAFIEVAAG, UGAROVATY, CHERNOV BK, SHATSKYIN: A C o m p l e x RNA S e q u e n c e Determines the Internal Initiation o f E n c e p h a l o m y o c a r d i t i s Virus RNA T r a n s l a t i o n . Nucl Acids Res 1990, 19:665-671.
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DUKEGM, HOFFMAN MA, PALMENBERGAC: S e q u e n c e a n d S t r u c t u r a l E l e m e n t s t h a t c o n t r i b u t e to E f f i c i e n t Enc e p h a l o m y o c a r d i t i s Viral RNA Translation. J Virol 1992, 66:1602-1609.
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NICHOLSONR, PELLETIERJ, LE SY, SONENBERG N: Structural a n d F u n c t i o n a l Analysis o f t h e R i b o s o m e L a n d i n g Pad o f Poliovirus Type: I n Vivo T r a n s l a t i o n Studies. J Virol 1991, 65:5886-5894.
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PILIPENKOEV, GMVLAP, MASLOVASV, SVlTKINYV, SINYAKOV AN, AGOL V: Prokaryotic-like Cis E l e m e n t s i n t h e CapI n d e p e n d e n t I n t e r n a l Initiation o f T r a n s l a t i o n o n Pic o r n a v i r u s RNA. Cell 1992, 68:119-131.
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MEEROV1TCH K, PELLETIER J, SONENBERG N: A Cellular P r o t e i n t h a t Binds to t h e 5'-Noncoding R e g i o n o f Poliovirus RNA~ I m p l i c a t i o n s f o r I n t e r n a l T r a n s l a t i o n Initiation. Genes Dev 1989, 3:1026-1034
14
KUHNR, LUZ N, BECKE: F u n c t i o n a l Analysis o f t h e Intern a l T r a n s l a t i o n Site o f F o o t - a n d - m o u t h D i s e a s e Virus. J Virol 1990, 64:4625-4631.
15.
BELSHAMGJ, BRANGWYNJK: A R e g i o n o f t h e 5' Nonc o d i n g R e g i o n o f F o o t - a n d - M o u t h Disease Virus Directs E f f i c i e n t I n t e r n a l I n i t i a t i o n o f P r o t e i n S y n t h e s i s w i t h i n Cells: I n v o l v e m e n t w i t h t h e R o l e o f L P r o t e a s e i n T r a n s l a t i o n a l Control. J Virol 1990, 64:5389-5395.
16.
BORMANA, JACKSON RJ: I n i t i a t i o n o f T r a n s l a t i o n o f Hum a n R h i n o v i r u s RNA: M a p p i n g t h e I n t e r n a l R i b o s o m e E n t r y Site. Virology 1992, 188:685-686.
17.
MACEJAKDG, SARNOWP: I n t e r n a l Initiation o f Translat i o n M~ediated b y t h e 5" Leader o f a Cellular mRNA. Nature 1991, 353:90-94.
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AGOLVI: T h e 5 ' - U n t r a n s l a t e d R e g i o n o f P i c o r n a v i r a l G e n o m e s . A d v Virus Res 1991, 40:103-180.
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MILLERAD, ROSMAN GJ: I m p r o v e d Retroviral V e c t o r s for G e n e T r a n s f e r a n d E x p r e s s i o n . Biotechniques 1989, 7:980-990.
Conclusion The advances in the field of picornavims translation have not only brought rich rewards in the understanding of viral reproduction, but h a v e also increased our knowledge of the eukaryotic translational apparatus. We can expect that more e x a m p l e s of internal initiation will be found in cellular mRNAs. O n c o g e n e s are possible candidates as their 5' UTRs are very long and contain m a n y AUG triplets. As the knowledge of picornavirus translation has o p e n e d n e w avenues in the engineering of m a m m a l i a n expression vectors, the study of IRES-directed translation in stable cell lines might shed light on the factors required for efficient initiation at the IRES. For example, h o w does the modification of initiation factors, brought about by serum deprivation, heat shock or mitosis, affect internal initiation c o m p a r e d to c a p - d e p e n d e n t translation? A better understanding of the interaction between cis-elements of the IRES and protein trans-factors is n e e d e d to clarify the process of internal ribosomebinding in eukaryotic mRNAs further.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1. 2.
KOZAKM: T h e S c a n n i n g M o d e l f o r Translation: A n Update. J Cell Biol 1989, 108:229-241. DONAHUETF: S c a n n i n g , I n t e r n a l Initiation a n d t h e Control o f t h e I n i t i a t i o n o f P r o t e i n Synthesis. Curr Opin Cell Biol 1990, 2:1087-1091.
3.
JANG SK, KRAUSSLICH HG, NICKLIN MJH, DUKE GM, PALMENBERG AC, WIMMER E: A S e g m e n t o f t h e 5' N o n t r a n s l a t e d R e g i o n o f E n c e p h a l o m y o c a r d i t i s Virus RNA Directs I n t e r n a l E n t r y o f R i b o s o m e s D u r i n g I n Vitro Translation. J Virol 1988, 62:2636-2643.
4.
JANG SK, DAVIESMV, I(AUFMANRJ, WIMMERE: I n i t i a t i o n of P r o t e i n S y n t h e s i s b y i n t e r n a l E n t r y o f R i b o s o m e s into t h e 5" N o n t r a n s l a t e d R e g i o n o f E n c e p h a l o m y o c a r d i t i s V i r u s R N A I n Vivo. J Virol 1989, 63:1651-1660.
20. •.
ADAMMA, RAMESHN, MILLERAD, OSBORNE WRA: I n t e r n a l Initiation o f T r a n s l a t i o n i n Retroviral Vectors Carryi n g P i c o r n a v i r u s 5' N o n t r a n s l a t e d R e g i o n s . J Viro11991, 65:4985-4990. This article demonstrates that retroviral vectors containing a poliovirus IRES can express two genes from a single transcript in a stable cell line, and that the 5' UTR poliovirus sequence does not interfere with the production of high virus titer. 21. •.
GHATrAS IR, SANES JR, MAJORS JE: T h e E n e e p h a l o m y o carditis Virus I n t e r n a l RtlJosome E n t r y Site Allows Efficient Coexpression of Two Genes from a Recombi-
Internal translation initiation in the design o f improved expression v e c t o r s Davies and Kaufman 5 1 7 nant P r o v i r u s i n C u l t u r e d Cells a n d in E m b r y o s . Mol
30.
Cell Biol 1991, 11:5848-5859. The use of the EMCV IRES in an RSV-derived vector is s h o w n to provide the most efficient means to coexpress two genes from a single provirus. Vectors encoding the lacZ gene and an IRES-v-src cassette were used in the generation of transgenic cells which can be identified with a histochemical stain and studied for their phenotype due to v-src expression.
KAUFMANRJ: T r a n s l a t i o n a l Efficiency o f P o l y c i s t r o n i c mRNAs a n d t h e i r Utilization to Express Heterologous Genes i n M a m m a l i a n Cells. EMBO J 1987, 6:187-193.
31. •.
KAUFMANRJ, DAVIES MV, WASLEY LC, MICHNICK D: Iln-
22.
K o o HM, BROWN AMC, KAUFMANRJ, PROROCK CM, RON Y, DOUGHERTYJP: A Spleen Necrosis V i r u s - b a s e d R e t r o v i r a l Vector w h i c h E x p r e s s e s T w o G e n e s f r o m a D i c i s t r o n i c MRNA. Virology 1992, 186:669-675. Another example of the use of the EMCV IRES in a retroviral vector. 23. •.
MORGANRA, COUTURE L, ELROY-STEIN O, RAGHEB J, Moss B, ANDERSON "WE: R e t r o v i r a l Vectors C o n t a i n i n g Putative Internal Ribosome Entry Sites: D e v e l o p m e n t o f a P o l y e i s t r o n i c G e n e T r a n s f e r System and AppHcat i o u s to H u m n n G e n e T h e r a p y . Nucl Acids Res 1992, 20:1293-1299. First example of the use of multiple IRES in retroviral vectors. Stable producer cell lines were established with tricistronic vectors, and were s h o w n to synthesize all three gene products and to produce high virus titer that could productively transduce 3T3 cells. 24.
FUERSTTR, NILES EG, STUDIER FW, MOSS B: E u k a r y o t i e
Transient-expression System Based o n R e c o m b i n a n t Vaccinia V i r u s t h a t S y n t h e s i z e s B a c t e r i o p h a g e T7 RNA P o l y m e r a s e . Proc Natl Acad Sci USA 1986, 83:8122~8126. 25.
ELROY-STEINO, FUERST TR, MOSS B: Cap-Independent T r a n s l a t i o n o f MILNA C o n f e r r e d b y E n c e p h a l o m y o carditis V i r u s 5' S e q u e n c e I x n p r o v e s t h e Performance o f t h e Vaceinia V i r u s / B a c t e r i o p h a g e T7 H y b r i d Expression S y s t e m . Proc Natl Acad Sci USA 1989, 86:612645130.
26. .,
MOLLAA, JANG SK, PAUL AV, REUER Q, WIMMERE: Cardiovir a l I n t e r n a l R i b o s o m a l E n t r y Site is F u n c t i o n a l i n a Genetically E n g i n e e r e d D i c i s t r o n i e Poliovirus. Nature 1992, 356:255-257. Insertion of the EMCV IRES into the ORF of the poliovirus polyprorein created a functional dicistronic poliovirus. This w o r k provides unambiguous evidence that the EMCV IRES can initiate translation internally in vivo. It also provides a strategy to analyze the processing of viral polyproteins or the rote of various domains in a polypeptide. 27.
KAUFMAN RJ:
Expression
Strategies for in Mnmmnllan
O b t a i n i n g H i g h Level Cells. Technique 1990,
2:221--236. 28.
WIGLERM. SWEET R, SIM GK, WOLD B, PELLICER A, LACY E, MANIATIST, SILVERSTEINS, AXEL R: T r a n s f o r t n a t i o n o f Mammallaq Ceils w i t h G e n e s f r o m P r o M o t e s and E u k a r y o t e s . Cell 1979, 16:777-785.
29.
KAUFMANRJ: Selection and C o a m p l i f i c a t i o n o f Heterologous Genes i n M a m m a l i a n Cells. Methods in Enzymol 1990, 185:537-566.
proved Vectors for Stable Expression o f Foreign Genes i n M a m m a l i a n Cells b y u s e o f t h e Untranslated
Leader S e q u e n c e f r o m EMC Virus. Nucl Acids Res 1991, 16:4485~i490. A family of dicistronic expression vectors containing the EMCV IRES is described. Efficient expression of the gene of interest is obtained from the adenovirus major late promoter and the expression of the selectable markers (DHFR and DHFR-mtxr), positioned downstream of the EMCV IRES, is independent from the upstream ORF. These vectors are suitable for transient or stable expression of foreign genes in mammalian cells. They yield high transformation efficiencies, are readily amplifiable and are less likely to rearrange or delete the gene of interest. 32.
DAVIESMD, KAUFMAN RJ: T h e S e q u e n c e C o n t e x t o f t h e I n i t i a t i o n C o d o n i n t h e E n e e p h a l o m y o c a r d i t i s Virus Leader Modulates Efficiency o f I n t e r n a l T r a n s l a t i o n Initiation. J Virol 1992, 66:1924-1932. This article studies the effect of the sequence context on translation initiation promoted by the EMCV IRES. Initiation was s h o w n to occur mainly at EMCV AUG-11 but is leaky. The sequence context downstream of EMCV AUG-11 influences the efficiency of initiation at that site as well as that of downstream AUGs. A purine at position - 3 is desirable, as in cap-dependent initiation. KIM DG, KANG HM, JANG SK, SHIN MS: Construction o f a B i f u n c t i o n a l mRNA i n t h e M o u s e b y Using t h e Intern a l R i b o s o m a l E n t r y Site o f t h e E n c e p h a l o m y o c a r d i t i s Virus. Mol Cell Biol 1992, 12:3636-3643. A dicistronic mRNA is s h o w n to be functional in mouse ES cells as well as in the chimeric embryos derived from them. This approach could be used to produce transgenic mice in which two genes can be expressed coordinately. 33.
34. •.
W O O D CR, MORmS GE, ALDERMAN EM, FOUSER L, KAUFMAN RJ: A n I n t e r n a l R i b o s o m e B i n d i n g Site can b e Used to Select for H o m o l o g o u s Recombinants at art Inlm u n o g l o b n l i n H e a v y - c h a i n Locus. Proc Natl Acad Sci USA 1991, 88:8006-8010. A sequence replacement-type vector containing a h u m a n T1 constant region sequence followed by an EMCV IRES-neo cassette is described. Flanking sequences homologous to murine genomic regions target that vector to the immunoglobulin heavy-chain locus. The strategy of using an EMCV IRES-neo cassette has potential application to a range of gene targeting vectors.
MV Davies and RJ Kaufman, Genetics Institute, 87 Cambridge Park Drive, Cambridge, Massachusetts, MA 02140, USA.
