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.     

Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation.

A comparison of picornavirus internal ribosome entry site (IRES) secondary structures revealed the existence of conserved motifs located on loops. We have carried out a mutational analysis to test their requirement for IRES-driven translation. The GUAA sequence, located in the aphthovirus 3A loop, did not tolerate substitutions that disrupt the GNRA motif. Interestingly, this motif was found at similar positions in all picornavirus IRESs, suggesting that it may form part of a tertiary-structure element. The RAAA tetranucleotide located in the 3B loop was conserved only in cardiovirus and aphthovirus. A mutational analysis of the RAAA motif revealed that activities of 3B loop mutants correlated with both the presence of a sequence close to CAAA at the new 3B loop and the absence of reorganization of the 3B and 3C stem-loops. In support of this conclusion, insertion of a large number of nucleotides close to the 3B loop, which was predicted to reorganize the 3B-3C stem-loop structure, led to defective IRES elements. We conclude that the aphthovirus IRES loops located at the most distal part of domain 3, which carries GNRA and RAAA motifs, are essential for IRES function.     

Structural organization of a viral IRES depends on the integrity of the GNRA motif

Little is known about the tertiary structure of internal ribosome entry site (IRES) elements. The central domain of foot-and-mouth disease (FMDV) IRES, named 3 or I, contains a conserved GNRA motif, essential for IRES activity. We have combined functional analysis with RNA probing to define its structural organization. We have found that a UNCG motif does not functionally substitute the GNRA motif; moreover, binding of synthetic GNRA stem-loops to domain 3 was significantly reduced in RNAs bearing UCCG or GUAG substitutions. The apical region of domain 3 consists of a four-way junction where residues of the GNRA tetraloop are responsible for the organization of the adjacent stem-loops, as deduced from ribonucleases and dimethyl sulfate accessibility. A single A-to-G substitution in the fourth position of this motif led to a strong RNA reorganization, affecting several nucleotides away in the secondary structure of domain 3. The study of mutants bearing UNCG or GUAG tetraloops revealed lack of protection to chemical attack in native RNA at specific nucleotides relative to the parental GUAA, suggesting that the GNRA motif dictates the organization and stability of domain 3. This effect is likely mediated by the interaction with distant residues. Therefore, the GNRA motif plays a crucial role in the organization of IRES structure with important consequences on activity.     

Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated

Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.     

Terminal RNA replication elements in human parechovirus 1

To define structural elements critical for RNA replication in human parechovirus 1 (HPeV1), a replicon with chloramphenicol acetyltransferase as a reporter gene and an infectious virus cDNA clone have been used. It was observed that there are cis-acting signals required for HPeV1 replication located within the 5'-terminal 112 nucleotides of the genome and that these include two terminal stem-loops, SL-A and SL-B, together with a pseudoknot element. Significant disruption of any of these structures impaired both RNA replication and virus growth. In view of the similarity in terminal structures to several picornaviruses, such as cardioviruses and hepatoviruses, the insights generated in this work are of wider significance for understanding picornavirus replication.     

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.     

RNA-protein interactions in regulation of picornavirus RNA translation.

The translation of picornavirus RNA occurs by a cap-independent mechanism directed by a region of about 450 nucleotides from the 5' untranslated region, termed an internal ribosome entry site (IRES). Internal initiation of protein synthesis occurs without any requirement for viral proteins. Furthermore, it is maintained when host cell protein synthesis is almost abolished. By using in vitro translation systems, two distinct families of IRES elements which have very different predicted RNA secondary structures have been defined. The cardiovirus and aphthovirus elements function very efficiently in rabbit reticulocyte lysate, whereas the enterovirus and rhinovirus elements function poorly in this system. However, supplementation of this translation system with additional cellular proteins can stimulate translation directed by the enterovirus and rhinovirus RNAs and reduce production of aberrant initiation products. The characterization of cellular proteins interacting with the picornavirus IRES is a major focus of research. Many different protein species can be observed to interact with regions of the IRES by in vitro analyses, e.g., UV cross-linking. However, the function and significance of many of these interactions are not always known. For two proteins, La and the polypyrimidine tract-binding protein, evidence has been obtained for a functional role of their interaction with IRES elements.     

The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.     

Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.     

Functional and structural similarities between the internal ribosome entry sites of hepatitis C virus and porcine teschovirus, a picornavirus

Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5' untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA(i)(Met). Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.     

A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae

The 5'-untranslated regions (5' UTRs) of picornavirus genomes contain an internal ribosomal entry site (IRES) that promotes the end-independent initiation of translation. Picornavirus IRESs are classified into four structurally distinct groups, each with different initiation factor requirements. Here, we identify a fifth IRES class in members of Kobuvirus, Salivirus, and Paraturdivirus genera of Picornaviridae: Aichi virus (AV), bovine kobuvirus (BKV), canine kobuvirus (CKoV), mouse kobuvirus (MKoV), sheep kobuvirus (SKV), salivirus A (SV-A), turdivirus 2 (TV2), and TV3. The 410-nucleotide (nt)-long AV IRES comprises four domains (I to L), including a hairpin (L) that overlaps a Yn-Xm-AUG (pyrimidine tract/spacer/initiation codon) motif. SV-A, CKoV, and MKoV also contain these four domains, whereas BKV, SKV, and TV2/TV3 5' UTRs contain domains that are related to domain I and equivalent to domains J and K but lack an AV-like domain L. These IRESs are located at different relative positions between a conserved 5'-terminal origin of replication and divergent coding sequences. Elements in these IRESs also occur elsewhere: domain J's apical subdomain, which contains a GNRA tetraloop, matches an element in type 1 IRESs, and eIF4G-binding motifs in domain K and in type 2 IRESs are identical. Other elements are unique, and their presence leads to unique initiation factor requirements. In vitro reconstitution experiments showed that like AV, but in contrast to other currently characterized IRESs, SV-A requires the DExH-box protein DHX29 during initiation, which likely ensures that the initiation codon sequestered in domain L is properly accommodated in the ribosomal mRNA-binding cleft.     

Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.     

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.     

Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.     

Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.

The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.     

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 properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon.

The internal ribosome entry segment (IRES) of picornaviruses consists of approximately 450 nt of 5'-untranslated region, terminating at the 3' end with an approximately 25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and finally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in encephalomyocarditis virus (EMCV) virtually all ribosomes initiate translation at this site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3' end of the IRES (at nt 586 in PV type 1) is considered to be a silent entry site, with all ribosomes initiating translation at the next AUG downstream (nt 743). To investigate what determines this different behavior, chimeras were constructed with a crossover at the conserved UUUC motif: the body of the IRES, the sequences upstream of this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of the FMDV or PV IRESes was replaced by that of EMCV, there was a marked increase in the absolute and relative frequency of initiation at the upstream AUG, the Lab site of FMDV and 586AUG of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, initiation occurred with no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the context of the AUG at the 3' end of the IRES may influence initiation frequency at this site, as was shown by improving the context of 586AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-competent manner is particularly efficient and accurate with the EMCV IRES.     

Polypyrimidine Tract Binding Protein-1 (PTB1) Is a Determinant of the Tissue and Host Tropism of a Human Rhinovirus/Poliovirus Chimera PV1(RIPO)

The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)''s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the functional IRES structure.