A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.     

Construction of regulatable picornavirus IRESes as a test of current models of the mechanism of internal translation initiation

Picornavirus internal ribosome entry sites (IRESs) are approximately 450 nt. RNA elements that direct internal initiation of translation, such that when placed between the two cistrons of a dicistronic construct, they drive independent translation of the downstream cistron. Consequently they have been widely used for coordinated expression of two or more proteins. All picornavirus IRESs have an AUG triplet at the very 3' end, which is thought to be the actual site of internal ribosome entry. However with some IRESs, such as foot-and-mouth disease virus, and especially poliovirus, the majority of ribosomes do not initiate translation at this putative entry site AUG, but at the next AUG further downstream, which is thought to be accessed by a process of linear ribosome scanning from the entry site. If this is so, then it should be possible to regulate IRES-dependent translation by inserting an iron responsive element (IRE) between the putative entry site AUG and the main functional initiation site. This should make IRES-dependent translation sensitive to the concentration of iron regulatory protein (IRP), the protein that specifically binds to the IRE. This has been attempted with both the foot-and-mouth disease virus and poliovirus IRESs, and was successful in so far as an inhibition specifically of IRES-dependent translation was observed that was strictly dependent on both the presence of IRP and of a functional IRE motif inserted in the sense orientation. However, the range over which expression could be varied was rather limited (three- to fourfold maximum), because some IRES-dependent translation remained completely refractory to inhibition by even very high IRP concentrations. In contrast, with a cap-proximal IRE in the 5' untranslated region of an mRNA translated by the scanning mechanism, addition of sufficient IRP results in complete inhibition. These results support the model of IRES-promoted ribosome entry at an upstream site followed by strictly linear scanning to the main functional initiation site for the majority of internal initiation events, but imply that some ribosomes must access the functional initiation site by another route, possibly a nonlinear shunting-like mechanism.     

Attenuation of Theiler's murine encephalomyelitis virus by modifications of the oligopyrimidine/AUG tandem, a host-dependent translational cis element.

A set of Theiler's murine encephalomyelitis virus mutants with engineered alterations in the conserved oligopyrimidine/AUG tandem (E. V. Pilipenko, A. P. Gmyl, S. V. Maslova, G. A. Belov, A. N. Sinyakov, M. Huang, T. D. K. Brown, and V. I. Agol, J. Mol. Biol. 241:398-414, 1994) were assayed for their growth potential in BHK-21 cells (as reflected in plaque size) and for neurovirulence upon intracerebral inoculation of mice. Tandem-destroying mutations, which included substitutions in the oligopyrimidine moiety and extended insertions into the oligopyrimidine/AUG spacer, exerted relatively little effect on the plaque size but ensured a high level of attenuation. The attenuated mutants exhibited remarkable genetic stability upon growth in BHK-21 cells. However, the brains of rare animals that developed symptoms after the inoculation with high doses of these mutants invariably contained pseudorevertants with the oligopyrimidine/AUG tandem restored by diverse deletions or an AUG-generating point mutation. The AUG moiety of the tandem in the revertant genomes was represented by either a cryptic codon or initiator codon. The results demonstrate that the tandem, while dispensable for the Theiler's murine encephalomyelitis virus growth in BHK-21 cells, is essential for neurovirulence in mice. Thus, the oligopyrimidine/AUG tandem is a host-dependent cis-acting control element that may be essential for virus replication under certain conditions. The functional activity of the tandem was retained when its oligopyrimidine or AUG moieties were made double stranded. A possible role of the tandem in the cap-independent internal initiation of translation on the picornavirus RNA templates is discussed.     

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5'' untranslated region.

Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.     

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.     

cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.     

Exploring IRES region accessibility by interference of foot-and-mouth disease virus infectivity

Translation initiation of picornavirus RNA is driven by an internal ribosome entry site (IRES) element located upstream of the initiator codon. RNA structure organization as well as RNA-protein interaction plays a fundamental role in internal initiation. IRES activity has been mainly analyzed in the context of reporter genes, lacking regions of the viral genome potentially affecting translation efficiency. With the aim to understand the vulnerability of the IRES and translation start region to small molecules in the context of the viral genome, we designed a set of customized RNase-resistant 2'O-methyl antisense oligoribonucleotides (2'OMe AONs) based on RNA structure data. These AONs were then used to monitor their capacity to interfere viral RNA translation, and thus, to inhibit virus yield. Foot-and-mouth disease virus (FMDV) RNA translation can be initiated at two in-frame AUG codons. We show here that a 2'OMe AON complementary to AUG2 inhibited viral multiplication more efficiently than the one that targeted AUG1. Furthermore, the response of the viral RNA to AONs targeting the IRES region denoted important differences between tissue culture cells and cell-free systems, reinforcing the need to analyze viral RNA response in living cells. Importantly, we have identified four specific motifs within the IRES element that are targets for viral inhibitors both in tissue culture cells and in cell-free systems. The identified targets define accessible regions to small molecules, which disturb either the RNA structural organization or the RNA-protein interactions needed to initiate translation in FMDV RNA.     

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.     

A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.     

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 5' noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.     

Unique characteristics of a picornavirus internal ribosome entry site from the porcine teschovirus-1 talfan

The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3' end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.     

Non-AUG-initiated internal translation of the L* protein of Theiler's virus and importance of this protein for viral persistence

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either acute encephalitis or persistent demyelinating disease. Persistent strains of Theiler's virus (such as DA) produce an 18-kDa protein called L* from an open reading frame overlapping that encoding the viral polyprotein. Neurovirulent strains (such as GDVII) are thought not to produce the L* protein, as the alternative open reading frame of these strains starts with an ACG codon instead of an AUG codon. However, we observed that both persistent and neurovirulent strain derivatives can produce two forms of the L* protein through unusual type II internal ribosome entry site-mediated translation. A full-length 18-kDa protein can be expressed from an ACG or an AUG initiation codon, whereas an N-terminally truncated 15-kDa product can be translated from a downstream AUG initiation codon. The expression of the 18-kDa form is required for efficient persistence of DA virus derivatives in the central nervous system.     

Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation

Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5' cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.     

Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.     

A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.