Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.

1607252

Enzyme 1990;44:292-309

Cap-Independent Translation of Picornavirus RN As: Structure and Function of the Internal Ribosomal Entry Site Sung Key Jang, Tatyana V. Pestova, Christopher U. T. Hellen, Gary W. Witherell, Eckard Wimmer Department of Microbiology, School of Medicine, State University of New York at Stony Brook, N.Y., USA

Key Words. Internal initiation • mRNA secondary structures ■ Host translational shut-off • mRNA-binding protein ■ Oligopyrimidine ■ Poliovirus • Human rhinovirus • Encephalomyocarditis virus • Foot-and-mouth disease virus • Hepatitis A virus

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Abstract. Picomaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differ ences among them in events leading to the initiation of protein synthesis. A large segment of the 5' nontranslated region, approximately 400 nucleotides in length, promotes ‘internal’ entry of ribosomes independent of the non-capped 5' end of the mRNA. This segment, which we have called the internal ribosome entry site (1RES), maps approximately 200 nt down stream from the 5' end and is highly structured. 1RES elements of different picomaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, 1RES elements of the genera entero- and rhinoviruses, on the one hand, and car dio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All 1RES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for 1RES function. The 1RES elements of cardio-, enteroand aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the 1RES is essential for translation in vitro. The 1RES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown. The function of p57 or p52 in cellular metabolism is unknown. Since picornaviral 1RES elements function in vivo in the absence of any viral gene products, we speculate that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation. 1RES elements are useful tools in the con struction of high yield expression vectors, or for tagging cellular genetic elements.

Introduction Members of the family Picornaviridae are prototypes of plus-strand animal RNA vi ruses, a group of viruses whose genomic RNAs serve as mRNA [1]. After entry into and uncoating inside the host cell, the viral genomic RNAs immediately engage in pro tein synthesis. The structure of the viral ge nomes was therefore thought to harbor all features of a mammalian mRNA, a hypothe sis that turned out not to be true [reviewed by Wimmer, 2], Similarly, it was assumed that the individual steps leading to the initia tion of protein synthesis are the same for cel lular mRNA and picornavirus genomic RNA. Our recent studies [3-5; Pestova et al., submitted for publication], and those of Sonenberg et al. [this volume], have shown that this assumption was also wrong. Instead, available evidence suggests that picornavirus mRNAs have adopted a mechanism of ini tiation of protein synthesis distinct from that used by most capped mRNAs. Picornaviridae are divided into the gen era Enterovirus (polio-, coxsackie- and echoviruses), Rhinovirus [human rhinoviruses (HRV)], Cardiovirus [encephalomyocarditis virus (EMCV), Theiler’s virus] and Aphthovirus [foot-and-mouth disease virus, (FMDV)]. A fifth genus, not yet officially assigned, will include hepatitis A virus (HAV) [6], The molecular biology of replica tion of these viruses is similar. A hallmark is the synthesis of a single gene product, the polyprotein, that is proteolytically cleaved to functional polypeptides [7], Thus, the ge netic structure of the picornavirus genome consists of a long 5' nontranslated region (5'NTR), followed by a single, long, open reading frame (between 2,332 and 2,178 co dons), a relatively short 3'NTR and a

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poly(A) tail [8, 9]. All picornavirus genomes are linked at their 5' end to a small protein, called VPg, that is removed from the 5'-ter minal uridylic acid nucleotide prior to the engagement of the genome in protein synthe sis. The absence of VPg does not diminish the infectivity of the viral RNA. This has been demonstrated most convincingly by the transcription of a poliovirus-specific cDNA clone with T7 RNA polymerase in vitro which yielded highly infectious RNA [10]. The mechanism of initiation of protein synthesis programmed by mammalian mRNA has been studied in detail. The model of ribosomal scanning [11] best fits the observed data, namely that the 5'-termi nal ‘cap’ (7mGpppG) of mRNA forms a complex with eukaryotic initiation factor eIF-4F. eIF-4F (itself a complex of 3 poly peptides), together with eIF-4A and eIF-4B, unwinds 5'NTR secondary structures and, by a mechanism yet to be determined, facili tates subsequent interaction of mRNA with the 40S small ribosomal subunit. The latter, in combination with factors like the RNA helicase eIF-4A, then ‘scans’ the 5'NTR of the mRNA to find the first, or sometimes the second or third initiation AUG codon, preferably in the nucleotide context of RXXAUGR (R = purine) [reviewed by Sonenberg, 12], Having selected the initiation codon, an initiation complex is formed and polypeptide synthesis commences. The car dinal rule of this model is that the ribosomes can enter the mammalian mRNA only via the capped 5' terminus. This explains a fun damental difference between gene expres sion by translation in pro- and eukaryotes: mRNAs of the former can be polycistronic (ribosomes can translate consecutive cistrons by entering the polynucleotide chain of the mRNA internally via the Shine-Dalgarno Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

Picornavirus Protein Synthesis

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Jang/Pestova/Hellen/Witherell/Wimmer


signal), whereas mRNAs of the latter are Discovery of Internal Ribosomal Entry monocistronic (ribosomes enter only from into EMCV mRNA the 5' end and, indeed, cellular mRNAs with consecutive cistrons have not yet been iden Picornavirus mRNA are, like their cellular counterparts, monocistronic as they express tified). Several observations concerning picor- only a single, long, open reading frame. This naviral mRNAs do not fit Kozak’s scanning property conforms to the structure and func model of mammalian cellular translation, tion of cellular mRNAs. However, the pe (i) The 5' end is not capped but instead is culiar features of picomavirus mRNA terminated with pUp... [13, 14], which is a prompted us to investigate whether its 5'-ter unique structure amongst mRNAs in mam minal pU can substitute for the capping malian cells, (ii) The 5'NTR is unusually group, or, more dramatically, whether the 5' long (between 624 and 1,199 nt). This defies terminus plays any role in translation at all. the notion that non-coding regions of viral A simple experiment was carried out: we RNA have evolved to be of minimal length synthesized a dicistronic mRNA in vitro (via because of the high mutation rate in genome T7 polymerase transcription of appropriate RNA replication and for reasons of genetic plasmids) and tested for the expression of economy [15, 16]. Since cis-acting signals for the second cistron when controlled by picorencapsidation or polymerase recognition are naviral 5'NTR, or derivatives thereof, by in likely to occupy no more than several dozens vitro translation in rabbit reticulocyte lysate of nucleotides, the conservation of the long (RRL). As a leader sequence for the control 5'NTR is indicative of an additional unique of the translation of the first cistron, we function, (iii) Preceding the codon at which selected the 5'NTR of poliovirus, because we polyprotein synthesis is initiated, picomavi- had observed earlier that poliovirus RNA is rus 5'NTRs contain up to 13 AUG triplets, an extremely poor mRNA in RRL [for refer none of which appear to be used in transla ences, see Nicklin et ah, 19]. We reasoned tion. (iv) Whereas small insertions in the that a second cistron following the poliovirus 5'NTRs of cellular mRNAs are not detri 5'NTR-controlled cistron should be ex mental to protein synthesis (since they do pressed very poorly, if at all, should ribo not interfere with scanning), similar modifi somes enter mRNA only at the 5' end. For cations in the 5'NTR of poliovirus [17], the control of the second cistron, we selected EMCV [Hoffman, Duke, and Palmenberg, the 5'NTR of EMCV mRNA since it has personal commun.; Witherell, Jang and been known for many years that this RNA is Wimmer, unpublished results] or FMDV a most efficient (possibly the most efficient) [18] can abolish mRNA function. This ob template in RRL [for references see Jackson, servation was interpreted to indicate the de 20], The dicistronic mRNA, therefore, had struction of structural elements in the picor- the structure (5' to 3'): poliovirus 5'NTR naviral 5'NTRs necessary for initiation of reporter protein X - EMCV 5'NTR - re protein synthesis. Surprisingly, the function porter protein Y (fig. 1A; note that the of the 5'NTRs in translation was sensitive to EMCV 5'NTR lacks the sequence preceding small modifications over a region of several and including the poly(C), as explained in the legend). hundred nucleotides.


5'

polk>

EMC

■[

3’

Ad

B

5 ,m76-/\/v-(

C

5’*7G-

EMC

X

ßGb

3’ EMC

X

[Y

3’

Fig. 1. Schematic representation of di- and tricistronic mRNAs whose cistrons are controlled by dif ferent 5'NTRs. Polio = The entire 5'NTR of poliovi rus; EMC = the 5'NTR of EMCV from nt 260 to the initiating AUG as depicted in figure 4A (the se quence preceding nt 260 contains a heteropolymeric segment and poly(C) which have no apparent in

fluence on the efficiency of translation); Ad and ßGb = the tripartite leader of late mRNA of adenovirus type 2, and the 5'NTR of ß-globin mRNA, respective ly. X, Y, and Z denote reporter gene. A Expression was tested in RRL and in a HeLa cell-free system. B, C Expression was tested in COS cells. For details see Jang et al. [3, 4],

The translation of the dicistronic mRNA (fig. 1A) in RRL gave the following results [3]: (i) the second cistron Y was expressed more efficiently than the first cistron; (ii) the second cistron Y was expressed at an earlier time than the first cistron, and (iii) deletions in the EMCV 5'NTR, controlling the second reporter protein, abolished expression of the second cistron Y. An explanation consistent with the data is that ribosomes entered the EMCV 5'NTR independently of the 5' end of the dicistronic mRNA. We have called the genetic element in the EMCV 5'NTR re sponsible for this phenomenon the ‘internal ribosomal entry site’, or 1RES [3, 4], We have tested the function of the 1RES in vivo by constructing vectors that, after trans fection into suitable cells, are transcribed to yield di- and tricistronic mRNAs (fig. lb) [4], Transfection of these vectors into suitable host cells produced the expected ‘internal’ gene product controlled by EMCV 5'NTR even when located as a third cistron Z, that is, several thousand nucleotides downstream from the 5' end. Again, a deletion in the EMCV 5'NTR of the polycistronic mRNA abolished this effect. To further substantiate

1RES function, we made use of the fact that poliovirus infection shuts off most cap-de pendent translation [21], An expression vec tor was constructed in which the first cistron was placed under the control of a cellular, capped leader, whereas the second cistron was controlled by the EMCV 1RES (fig. 1C). Superinfection with poliovirus of cells trans fected with the plasmid shown in figure 1C yielded a product only from the second, IRES-controlled cistron Y, but not from the first cistron X [4]. These data are only com patible with a mechanism of initiation of protein synthesis that is independent of the 5' end of the mRNA. Pelletier and Sonenberg [22], studying the 5'NTR of poliovirus RNA, have come to a similar conclusion.

Secondary Structures of the Picornavirus 5'NTRs The existence of higher order structures within the long 5'NTR was predicted as the subsequences of the viral RNAs were being elucidated, and, in one instance, biochemi cal data obtained during the course of the Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

A

295


sequence analysis of poliovirus RNA di rectly demonstrated a stable hairpin struc ture proximal to the 5' end [23]. Through careful analyses carried out by Pilipenko et al. [24, 25], the secondary structures of 5'NTRs of picornaviral RNAs have been elucidated by phylogenetic and biochemical studies. A structure for the 5'NTR of polio virus, very similar to that obtained by Pili penko et al. [24], was independently pro posed by Skinner et al. [26]. Inspection of the foldings led to the inter esting realization that the secondary struc tures of the picornaviral 5'NTRs can be di vided into two groups: that of the enteroand rhinoviruses (fig. 2), and that of cardio-, aphtho- and HAVs (fig. 3, note that HAV will be classified into a separate genus). Thus, the relationship of 5'NTR structures follows the genetic kinship of picomavirus genera as determined by alignment of nu cleotide sequences and calculation of a tree of maximal parsimony [27]. It is safe to pre dict that the function of the 1RES elements will parallel structural relatedness. Indeed, translation of viral RNAs in RRL already supports this notion: RNAs of EMCV and aphthoviruses are superb templates in RRL, whereas RNA of entero- and rhinoviruses are not (HAV, as in many aspects of its molecular biology, does not fit the rule) [6], In fact, poliovirus RNA yields aberrant translation products in RRL. This artifact

Fig. 2. Predicted folding patterns of portions of 5'NTRs of prototypes of the genera Entero- and Rhi novirus. A Poliovirus type 1 (Mahoney). B Human rhinovirus, type 14. The Yn-Xm-AUG units are indi cated by solid bar-gap-# symbols the initiating AUGs are marked by asterisks. The folding patterns are modifications of those proposed by Pilipenko et al. [35], For details and references, see text. Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

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can be eliminated if the RRL is supple mented with a HeLa cell extract [28]. Determination of the relationship of structure to function of picornavirus 5'NTR is currently pursued in many laboratories [see chapters in this volume by Sonenberg and Sarnow, and 18, 29-36, and references therein]. Our work has concentrated on the 1RES of EMCV and poliovirus; because of space limitations, we cannot review all the published papers. The reader is also referred to two other reviews by Agol [37] and Jackson et al. [38], Boundaries of the 1RES Element of EMCV Using dicistronic mRNA as diagrammed in figure 1 A, we performed a series of 5' 3' and 3' 5' deletion experiments within the 1RES element to determine the minimal length of 1RES that would still allow its func tion in RRL [5], Deletion up to nt 403 reduced the translational efficiency 5-fold, an observation for which we will offer an explanation below. Further deletion all but


Fig. 3. Predicted folding patterns of portions of 5'NTRs of prototypes of the genera Cardiovirus (A), Aphthovirus (B), and Hepatovirus, the latter being an unofficial term for a fifth genus of Picomaviridae that includes hepatitis A virus (C). The Yn-Xm-AUG units are indicated by solid bar-gap-asterisks the ini tiating AUGs are marked by asterisks. The patterns shown for EMCV and FMDV are modifications of those proposed by Pilipenko et al. [25]; that of HAV was derived from Hellen [unpublished data]; a very similar pattern has been proposed by Lemon et al. [personal commun.]. The properties and aspects of replication of HAV are clearly distinct form that of the rest of picomaviruses. Neverthless, the HAV 5'NTR resembles that of EMCV and FMDV in sev eral respects [Hellen and Wimmer, unpublished re sults; for review see Wimmer and Murdin, 6].


abolished translation of reporter protein Y. We have, therefore, assigned the 5' border of EMCV 1RES roughly to nucleotide 403, that is, just to the beginning of stem-loop E (fig. 4A). Similar deletions at the other side defined the 3' border roughly to nt 815, that is, at the 3' border of an oligopyrimidine (Yn) tract (fig. 4A). We have found that the deletions had the same effect on translational efficiency of monocistronic mRNA (with the structure EMCV 5'NTR-reporter protein Y), that is, the deletion of the E loop (up to nt 484) will reduce translation of reporter protein Y more than 100-fold even if this cistron is not pre ceded by the poorly translated, polio 5'NTRcontrolled first cistron [3, 5]. However, Kam inski et al. [32] have shown that a deletion up to nt 802 (that is, removal of most of the highly structured 5'NTR of EMCV RNA) leads to a partial revival of translation of monocistronic constructs. Translation of such extensively truncated RNAs is stimu lated by capping, an observation suggesting that EMCV RNA from which most of the 5'NTR has been removed begins to resemble cap-dependent cellular mRNA (see below). We believe, therefore, that the in vitro tests for 1RES function should be carried out in dicistronic mRNAs to reduce the possibility of 5'-terminal entry of ribosomes.

Binding of the Cellular Protein p57 to the Stem-Loop E of EMCV 5'NTR In light of the importance of stem-loop E for 1RES function, we entertained the possi bility that a specific cellular factor might bind to this structural element (fig. 4A). To investigate this possibility, synthetic, 32Plabeled RNA fragments were UV cross-

linked to proteins of crude RRL, or to pro teins of the ribosomal salt wash fraction. In this manner, we identified a cellular protein of 57 kD (p57) with specific affinity for stem-loop E [5]. Interestingly, the binding of p57 was abolished when the stem adjacent to loop E was destabilized by two point muta tions, and binding was re-established when the stem was regenerated by two compensa tory point mutations. Significantly, the bind ing of p57 correlated with 1RES function [5], We have therefore concluded that p57, whose cellular function is unknown, is a required factor in the initiation of transla tion of EMCV RNA in vitro. Whether this is true also in vivo remains to be seen. Shatsky and his colleagues have independently de tected a cellular protein (‘p58’) that binds to nt 315-485 of the 5'NTR of EMCV. It is very likely that the protein found by Jang and Wimmer [5] (p57) and Shatsky’s ‘p58’ are identical polypeptides. Luz and Beck [33] have reported the binding of a p57 protein to the 5'NTR of FMDV. Although the binding to a specific loop has not been determined, it appears that the binding occurs in a manner similar to EMCV 5'NTR. However, these authors reported that p57 can also bind to a sequence within the FMDV 5'NTR that is located around and contains the Yn tract of this RNA. Attempts to reproduce this result with RNA surrounding the Yn tract of the EMCV 5'NTR have failed [Jang and Wimmer, un published results] and cannot be explained at present. As mentioned above, deletion ofthe EMCV 5'NTR to the 5' boundary of 1RES reduced translational efficiency 5-fold [5]. Inspection of the deleted sequences revealed another stem-loop (‘C; fig. 4A) whose structure is re lated to stem-loop E. An RNA containing Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

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stem-loop C but not E can also bind p57, and this binding is reduced in competition exper iments with RNA corresponding to stem-loop E, but not with unrelated RNA [Witherell et al., unpublished results]. We do not yet know whether binding of p57 to the C loop corre lates with translational efficiency. We enter tain the possibility, however, that the pres ence of both loops in the 5'NTR enhances the binding of the cellular protein to the viral 5'NTR, possibly in a cooperative manner, thereby increasing internal ribosomal bind ing. This may contribute to the advantage EMCV RNA has over cellular mRNAs when they are co-translated in vitro [39].

Oligopyrimidine Tract in Picornaviral 5'NTRs The existence of an oligopyrimidine (Yn) tract near the initiating AUG was observed some time ago in FMDV RNA by Beck et al. [40], Its significance remained unclear until it was found that mutations within the Yn tract dramatically influenced the efficiency of translation of mRNA controlled by FMDV 5'NTR [18]. Similarly, we found that the deletion of a few of the 9 Y residues at the 3' border of EMCV 1RES abolished translation [5]. It is therefore evident that the Yn tract plays a role in the initiation of translation. The Yn tract in the 5'NTRs of EMCV and FMDV are 16-18 nt upstream of the initiat ing AUG codon (for EMCV see fig. 4A), and their function may be related to the selection of the correct AUG in polyprotein synthesis. There are, however, profound differences in the location of the Yn tract with respect to the initiating AUG codon, when different picornavirus 5'NTRs are inspected.

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A very short Yn tract can be found in pol iovirus type 1 (Mahoney) RNA near the ini tiating AUG codon [40], but this tract can not be important because it is not conserved amongst the other types of poliovirus [41], On the other hand, the 5'NTRs of all enteroand rhinoviruses contain a conserved Yn tract roughly between nt 560 and nt 578, which, in poliovirus type-1 RNA is located 154 nt upstream of the initiation codon (in rhinovirus type 14 the distance is 31 nt). Mutational analysis in the Yn tract of polio virus clearly indicated that this element is important in translation in HeLa cell ex tracts (see below). Closer inspection of the 1RES elements of picornaviruses, however, reveals an unex pected similarity with respect to the Yn tracts. In all cases, the Yn tracts are located 10-20 nt upstream from an AUG triplet. In polio- and rhinoviruses 5'NTRs, this AUG triplet (which is in poor nucleotide context) does not serve as an initiating codon in translation (attempts to identify a 6-kDa peptide specified by the downstream ORF of this AUG in poliovirus RNA have failed in different laboratories) [Dorner and Wim mer, unpublished results]. This AUG triplet must nevertheless play a role in replication since point mutations targeted to it yielded a poliovirus variant with small plaque pheno type [42], Meerovitch et al. [43] have re ported the binding of a cellular 52-kD pro tein to a sequence of the poliovirus 5'NTR (nt 559-624) harboring this AUG and also the Yn tract [see Sonenberg, this volume]. This sequence was originally reported by Bienkowska-Szewczyk and Ehrenfeld [44] to be important for translation of poliovirus RNA in vitro. Sonenberg [this volume] has observed that the addition of partially puri fied p52 to in vitro translation mixtures Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM


Fig. 4. (For legend see p. 301)

J ang/Pestova/Hellen/Witherell/Wimmer


300


301

c p

r

Poliovirus Type 1 (M)

C.C

U.R P.U R.U C.C

P.U C.ü U.P

JLU c c u p R U U u. p

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c

p

c u

fiC

C GUOUUUCCUUUURUUUURUUGUGG CCRPUUCGP

B

P l) P

I

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P U U P

RC U.R R.U U.R R.U C

5'

Type 14 P

C.C R.U C.C R.U C.G U.R C.U c C u R R U

*

3' u GG U GUtUUUCUCflUUUUUCUUCfiUflUUGUC GCUCfìGGUlllCUflCPCRGfìfìflfìG

Fig. 4. Detailed sequence folding patterns of por tions of the 5'NTRs of several picomaviruses. A Fold ing of EMCV 5'NTR between nt 260 and 890. The initiating AUG No. 11 is boxed in, other AUGs are indicated by pronounced roman numbers along the nucleotide sequence. The shaded areas depict binding of cellular proteins p57 to stem-loop E [5], and one or

more proteins (denoted ‘X’) to the Yn-Xm-AUG unit [Jang and Wimmer, unpublished; see text]. The con tact points between RNA and proteins are unknown. Since p57 is a ribosome-associated protein [5], it was drawn such that it contacts the 40S ribosomal subunit at the 3' border of the EMCV 1RES. B Poliovirus type 1 (Mahoney). C Rhinovirus type 14 (HRV14).

stimulates the translation of poliovirus RNA. We have investigated the importance of the primary structure of the 14-nt-long Yn tract in the poliovirus 5'NTR by introducing site-directed mutants throughout this

strongly conserved sequence (table 1) [Pestova et al., submitted for publication]. Sub stitutions within the 3' half of this tract (nt 564-569; mutants 5-7) had little or no effect, whereas all substitutions within the 5' half (nt 557-563; mutants 1-4) reduced the effiDownloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

c

Rhinovirus


302

Table 1. Effect of mutations in the polypyrimidine tract of the poliovirus 5'NTR on translation in vitro 560

Polio 1 (M); Mutant 1 Mutant 2 Mutant 3 Mutant 4 Mutant 5 Mutant 6 Mutant 7 Mutant 8

G U G U U . . . G A

570

U C C U U

U U A U U

U U A U U

Translation in vitrob

Q

++++++ ++

A A . G G

+

G G

..GA

+++

G G C C

G

G

C c C C

G G

G G

++++ ++++++

a Segment containing the oligopyrimidine tract of poliovirus type 1 (Mahoney). b Translation in a HeLa cell lysate of synthetic mRNA containing nt 70-743 of the poliovirus 5'NTR and the open reading frame for PI, the capsid precursor.

reduced affinity for p52 is probably not re sponsible for the reduced translational effi ciency of the mutants described above. The reduced translation efficiency of these mutants may therefore reflect unpredicted disruption of secondary or tertiary structures, reduced affinity for a protein that we have been unable to detect by UV cross-linking, or disruption of the potential interaction be tween the 3'-terminal region of 18S RNA and the oligopyrimidine tract (see below). Similar results have been obtained in mu tational analyses of the Yn tract proximal to the initiating AUG in the EMCV 5'NTR. Perturbation of this tract by insertional or deletional mutation drastically reduced 1RES function in dicistronic mRNA in vitro [Jang and Wimmer, unpublished results]. Addition of nucleotides before or after the Yn tract also reduced the translational efficiency about 10fold, an observation suggesting that the Yn tract may not work independently but as a part of a larger functional unit. Indeed, we believe that this unit is the conserved YnXm-AUG, where m is the number of random Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

ciency of translation of a reporter gene (the PI capsid protein precursor, naturally lo cated immediately downstream of the 5'NTR) in a cell-free extract of HeLa cells. The essential core of this domain comprises nt 559-561 (UCC), since substitution of these residues (mutants 2 and 3) virtually abolished translation. This is consistent with the effects of deletion within the 5'NTR on virus viability reported by Iizuka et al. [45]. The determinants of the interaction of the above mentioned cellular protein p52 with 5'NTR have not been precisely determined, and we therefore considered the possibility that substitutions between nt 557 and 563 reduced translational efficiency by reducing the affinity of p52 for the 5'NTR. However, using a UV cross-linking assay, we have found no difference between the amount of p52 (and other proteins) bound to an RNA segment (nt 542-629) containing either wt or mutated oligopyrimidine tracts. We there fore conclude that the oligopyrimidine tract is not a determinant of binding of p52 to the 5'NTR of poliovirus, and consequently that


nucleotides (X) separating Yn and AUG (m is 10-20 nt; see below). In our search for a cellular protein that may bind to the region preceding and includ ing the initiating AUG (designated as ‘X’ in fig. 4A), we performed UV cross-linking ex periments with 32P-labeled RNA probes spanning nt 782-841. For comparison, we used a poliovirus RNA probe of nt 555-629 which is similar to that used by Meerovitch et al. [43] in the detection of p52. Interest ingly, both EMCV and poliovirus RNA probes showed exactly the same patterns of UV cross-linked proteins from RRL, ribo somal salt fraction of RRL, or post-ribosomal fraction of RRL. A polypeptide with an apparent molecular weight of 52 kD (p52) bound to the probes and it was fractionated with post-ribosomal proteins [Jang et al., in preparation] which we believe corresponds to p52 described by Meerovitch et al. [43], Deletion of the initiating codon of EMCV RNA reduced binding of p52. Deletion of a part of Yn element in the EMCV 1RES, on the other hand, did not show much effect on binding of p52.

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sis. This observation strongly suggests that a process of scanning does not occur in the ini tiation of EMCV polyprotein synthesis; rath er, the 40S ribosomal subunit is directly transferred to the proper AUG No. 11 [5], Kaminski et al. [32], using derivatives of EMCV 5'NTR-reporter protein constructs of Jang and Wimmer [5], have carried out experiments providing direct evidence for this hypothesis. Briefly, Kaminski et al., [32] found that AUG No. 11 is the principal ini tiation codon used as long as the 1RES is intact. Deletion up to nt 484 (removal of the E loop) rendered the synthetic mRNA inac tive in RRL, regardless of whether it was capped or not. Deletion up to nt 737, on the other hand, allowed translation starting at AUG No. 10, and even at AUG No. 8 and AUG No. 9 located in the H loop (fig. 4A). Translation was now dependent on capping, with the possible involvement of scanning. Kaminski et al. [32] concluded that the EMCV 1RES renders the translation capindependent, and that a mechanism of scan ning is absent. It follows that the EMCV 1RES marks, by whatever mechanism, AUG No. 11 for the initiation of translation of the EMCV polyprotein.

Selection of the Polyprotein-Initiating AUG Codon in EMCV RNA Selection of the Initiating AUG Codon of Picornaviruses RNA: A Unifying Working Hypothesis If initiation of EMCV polyprotein synthe sis occurs by the direct, cap-independent rec ognition of the AUG codon of a ‘Yn-XmAUG’ unit (Y9-X18-AUG), does this mech anism operate in the initiation of translation of other picomavirus polyproteins as well? Depending on the picomavirus, the answer is partly yes, and partly no. Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

The codon initiating EMCV polyprotein synthesis (AUG No. 11) resides in a cluster of 4 closely spaced AUGs (fig. 4A). Two of these (No. 10 and No. 11) are in the highly favored RXXAUGR context. AUG codons No. 10 and No. 13 are out of frame with the polyprotein; the open reading frame of AUG No. 10 is terminated 35 nt codons down stream [46]. The startling fact is that al though AUG No. 10 is only 5 nt upstream of AUG No. 11, it is not used in protein synthe

We shall first consider translation of FMDV RNA whose 1RES within the 5'NTR probably has a structure resembling that of EMCV (fig. 3B) [25]. Not surprisingly, a YnXm-AUG unit can be found in the 3' portion of the FMDV 1RES. However, a second YnXm-AUG unit exists 81 nt downstream from the first element (counted from AUG to AUG; in some serotypes, the length of the distance between the AUGs is only 7 5 nt) [for details and references, see Sangar et al., 47], We will refer to these units (5" —> 3') as YnXm-AUGI and II that are separated by a ‘spacer’ (81 or 75 nt). The AUGs of I and II occur in frame, and the spacers never contain an extra AUG triplet. Whereas the sequences preceding Yn-Xm-AUGI (that is the se quence forming the suggested FMDV 1RES) are conserved amongst serotypes of FMDV, the sequences of the spacers in different FMDV types are highly heterologous [47], The startling observation is that in some serotypes the AUGs of both Yn-Xm-AUG I and II units are used for initiation of polypro tein synthesis in vivo and in vitro, whereas in others, only the AUG of the second unit is used. It appears that a major determinant in the selection of the initiating AUG codon is its context. The AUG codon of the first ele ment occurs in a poor context in most sero types, and polyprotein synthesis is therefore initiated more efficiently at the AUG of the downstream Yn-Xm-AUG II unit, which has a good context. Although initiation of trans lation at the first AUG occurs inefficiently, or not at all in some serotypes, it is notable, and possibly significant that other features of the first unit (such as the length and integrity of the Yn tract, and the length of the Xm) are highly conserved. In other words, Yn-XmAUG I may serve a function distinct from and in addition to initiation of polyprotein

J ang/Pestova/Hellen/W itherell/Wimmer

synthesis of some FMDV serotypes. If YnXm-AUG I is used for initiation of transla tion it is likely that, as in EMCV, its AUG codon is not selected by a scanning mecha nism. It is possible, however, the selection of the AUG codon of Yn-Xm-AUG II (which invariably occurs in good context, but is often preceded by a degenerate polypyrimid ine tract) involves local scanning from the 3' boundary of the 1RES. A related situation exists in entero- and rhinoviruses. The 5'NTRs of both genera contain a Yn-Xm-AUG unit at the 3' border of the 1RES, but the AUGs of these elements all occur in a poor context and are never used to initiate translation. This, and the strong conservation of other features of these units are reminiscent of Yn-Xm-AUG I of some serotypes of FMDV; moreover, it has also been suggested that selection of the bona fide initiating AUG codon in entero- and rhinoviruses involves local scanning from the 3' border of the 1RES [37, 38]. The initiating AUG codon is separated from the Yn-XmAUG unit by a spacer that ranges in length from 31 nt (rhinovirus type 14) to 154 nt (poliovirus). Although Beck et al. [40] noted a Yn tract near the initiating AUG codon of poliovirus type 1 (Mahoney), it is degenerate (UUAUUUCAAUC). Moreover, such Yn tracts are entirely absent near the initiating AUG codons in the 5'NTRs of other poliovi rus types, all other enteroviruses and all rhi noviruses. Since virtually all enteroviruses and rhinoviruses thus contain only the YnXm-AUG I unit, we conclude that Yn tracts preceding an initiating AUG codon are not required if initiation of protein synthesis oc curs downstream from the site of loading of the 40S ribosomal subunit. Considering the fact that the spacer se quences have highly diverged even amongst Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

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the different types of polioviruses, and that the spacer sequence is much shorter in the related 5'NTR of HRV14 when compared to poliovirus, Kuge and Nomoto [48] and Kuge et al. [49] reasoned that spacers in poliovirus 5'NTRs may not be essential for viral prolif eration. Accordingly, they deleted most of the spacer in the 5'NTR of poliovirus type 1 and obtained viable virus [48], Particularly interesting is the deletion from nt 564-726 that removes a portion of Yn-Xm-AUG I, but regenerates a similar unit with nucleo tides between nt 726 and 746 [37], The regen erated unit is not perfect, which may be the reason for the small plaque phenotype of the mutant. Kuge et al. [49] also tested whether the spacer could tolerate an insertion as long as 71 nt (a polylinker sequence). Incredibly, viable virus with no apparent phenotype was obtained as long as the inserted sequence lacked an AUG codon [49], Taking these observation into consider ation, we propose the following model of ini tiation of translation of picomavirus RNAs. (l)The Yn-Xm-AUG unit, which so far has been found in all picomavirus 5'NTRs, is an essential component of the picomavi rus 1RES (fig. 5). It is possible that the Yn tracts interact with oligopurine sequences at the 3' end of 18S ribosomal RNA to promote binding of the 40S ribosomal subunit [50, 51]. (2) Uniquely, the 5'NTR of EMCV con tains only Yn-Xm-AUG I, whose AUG is in good context and therefore used for initia tion of polyprotein synthesis. Spacer and downstream initiation site are absent (fig. 5 A). Initiation of EMCV protein synthe sis is therefore the least complicated, since the formation of the initiation complex oc curs directly at the border of the EMCV 1RES.

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(3) FMDV 5'NTR contains two Yn-XmAUG units (fig. 5B). The Yn tract of unit II in some serotypes is degenerate, or even par tially deleted (e.g. in serotype SAT-3), yet initiation takes place largely at the AUG of this unit. We propose that this may occur as a result of internal entry, involving Yn-XmAUG unit I, failure to initiate there because of poor context of the AUG, followed by scanning to the next good context AUG which happens to occur as a residual part of unit II. The AUG codons of both units are always in frame, and they are separated by a spacer sequence with an open reading frame (fig. 5B). Initiation at unit I leads to a larger ‘leader peptide’ of the polyprotein. As pointed out, in some serotypes both larger and smaller leader peptides are synthesized in vivo, although synthesis of the shorter variety is more common. (4) Entero- and rhinoviruses have gener ally only unit Yn-Xm-AUG I. However, the AUG of this unit is never used for initiation of protein synthesis. Initiation occurs at an AUG codon downstream from unit I, and it is separated by an unconserved, non-coding spacer sequence of varying length (31 nt for HRV14, fig. 5C, or 154nt for poliovirus, fig. 5D). (5) It is unknown why the insertion of spacers separating unit I and the initiating AUG codon has evolved for most picomavi rus 5'NTRs. It appears that the spacer of pol iovirus can be deleted by genetic engineering [48] which then results in an 1RES reminis cent of rhinovirus or even EMCV. Since AUG codons engineered into the spacer are detrimental to viral proliferation, but an in sertion to increase spacer length is not [49], transfer of the ribosomal subunit from the 1RES to the initiating AUG may occur by scanning. Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM


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(6) At least two cellular proteins have been identified that participate in 1RES function: p52 and p57. Protein p52 appears to be involved in the function of the Yn-XmAUG unit, whereas p57 may mediate bind ing of the ribosomal subunit to the 1RES. Protein p57 also binds to the poliovirus 1RES; the precise binding site has not been mapped [Pestova et al., unpublished obser vation], A stem-loop structure similar to stem-loop E of the EMCV 1RES does not appear to exist in the poliovirus 1RES. This dilemma may be solved once the exact con tact points between protein and RNA have been identified. Considering the novel con cept of RNA/protein interaction that is emerging form recent studies in several labo ratories [52], we may find that structure, rather than nucleotide sequence, is the pre dominant determinant for the binding of p57 to picomavirus 1RES. (7) It is likely that, in addition to p52 and p57, other cellular and even viral proteins may participate in 1RES function. Some of these factors may be ubiquitous in all mam malian cells, such as the well-known cellular initiation factors eIF-4A and eIF-4B, others may be specific for certain differentiated cells. This is underscored by the differential function of poliovirus mRNA in RRL, on

quently degenerate and probably not essential. AUG in an open box has the propensity to initiate polypro tein synthesis; AUG in the hatched box never func tions as an initiator codon. The curved lines between the two AUGS are the spacer sequences, whose lengths in nt is given in arabic numerals. The double arrows on this line indicate that scanning is likely to occur through this sequence if the first AUG is not used for initiation. Bold arrows indicate the initiation of polyprotein synthesis. For further details, see text. Downloaded by: University of Exeter 144.173.6.94 - 6/7/2020 7:49:29 PM

Fig. 5. Model for the selection of the initiating AUG in picomaviruses. Only the 1RES of EMCV is boxed in [5]. The borders of 1RES elements of other picomaviruses are not yet known and may vary when translation occurs in different host cells [37, 38]. Cel lular protein p57 binds to 1RES elements of EMCV, FMDV, and poliovirus, but the region to which it binds is only known for EMCV (stem-loop E) [5]. p52 appears bound to a portion of the Yn-Xm-AUG unit [Sonenberg, this volume]. Only FMDV has two YnXm-AUG units, but the Yn of the second unit is fre-


Application of 1RES Elements in Molecular Biology Apart from basic interest in the unique features of the initiation of translation of picomavirus mRNA, 1RES elements have been used successfully for the construction of vectors affecting cap-independent transla tion in mammalian cells. Although the liter ature dealing with this subject is as yet sparse, Elroy-Stein et al. [53] have used the EMCV 5'NTR under the control of the phage T7 promoter for high expression of heterologous proteins in their vaccinia virus system, which provides phage T7 RNA poly merase in trans. Similarly, plasmids contain ing the EMCV 5'NTR under the control of phage T3 promoter directed good expression of reporter genes in cells supplying T3 RNA polymerase [54], The 1RES of EMCV is a suitable leader for the development of a eukaryotic pro moter selection cassette by combining the EMCV 1RES with a selectable marker gene. As long as insertion occurred in exons, the selectable marker gene will be expressed re gardless of position or of reading frame within the target gene.

Acknowledgments We are indebted to Vadim Agol and Richard J. Jackson for discussions, James Harber and Kevin Harris for computer work, and Connie Rafferty for the preparation of the manuscript. This work was supported, in part, by Public Health Service grant AI15122 and Ca-28146 from the National Institutes of Health.

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B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression.

Beclin-1-independent autophagy positively regulates internal ribosomal entry site-dependent translation of hypoxia-inducible factor 1α under nutrient deprivation.

Understanding the potential of hepatitis C virus internal ribosome entry site domains to modulate translation initiation via their structure and function.

Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant.

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

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Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

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Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage.

A comparative study on translation of flavivirus and picornavirus RNAs in vitro: apparently different modes of protein synthesis.

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Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.

Oligonucleotide mapping of picornavirus RNAs by two-dimensional electrophoresis.

Ribosomal RNA and translation.