Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

Replication of poliovirus RNA with complete internal ribosome entry site deletions

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.     

IRES-mediated pathways to polysomes: nuclear versus cytoplasmic routes

Eukaryotic mRNA initiates translation by cap-dependent scanning, ribosome shunting and cap-independent internal ribosome entry. Internal ribosome entry was first discovered for cytoplasmic RNA viruses but has also been identified for DNA viruses and cellular mRNAs. An internal ribosome entry site (IRES) directs internal binding of ribosomes and nucleates the formation of a translation initiation complex. Current research is aimed at identifying interactions between IRES elements and RNA-binding proteins known as ITAFs (IRES trans-acting factors). Here we compare IRES elements from cytoplasmic RNA viruses with those of cellular mRNAs and DNA viruses with nuclear mRNA synthesis, and suggest that ITAF composition and IRES function directly reflect the site of synthesis of mRNA and the history of its pathway to polysomes.     

A search for structurally similar cellular internal ribosome entry sites

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function.     

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

Picornaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differences 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 (IRES), maps approximately 200 nt down-stream from the 5' end and is highly structured. IRES elements of different picornaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, IRES elements of the genera entero- and rhinoviruses, on the one hand, and cardio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for IRES function. The IRES elements of cardio-, entero- and aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IREs is essential for translation in vitro. The IRES 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 IRES 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. IRES elements are useful tools in the construction of high yield expression vectors, or for tagging cellular genetic elements.     

Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus.The dependence of viruses on the host translation system imposes constraints that are central to virus biology and have led to specialized mechanisms and intricate regulatory interactions. Failure to translate viral mRNAs and to modulate host mRNA translation would have catastrophic effects on virus replication, spread, and evolution. Accordingly, a wide assortment of virus-encoded functions is dedicated to commandeering and controlling the cellular translation apparatus. Viral strategies to dominate the host translation machinery target the initiation, elongation, and termination steps and include mechanisms ranging from the manipulation of key eukaryotic translation factors to the evolution of specialized cis-acting elements that recruit ribosomes or modify genome-coding capacity. Because many of these strategies have likely been pirated from their hosts and because virus genetic systems can be manipulated with relative ease, the study of viruses has been a preeminent source of information on the mechanism and regulation of the protein synthesis machinery. In this article, we focus on select viruses that infect mammalian or plant cells and review the mechanisms they use to exploit and control the cellular protein synthesis machinery.     

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 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.     

RNA病毒翻译调控元件—内部核糖体进入位点(IRES)

真核生物大多数蛋白质合成采用了依赖帽子结构的翻译起始方式.但一组缺乏帽子构的RNA病毒的蛋白质合成起始是依赖其5′端非翻译区（untranslated region,UTR）翻译调控的顺式作用元件——内部核糖体进入位点（internal ribosome entry site, IRES）.它 们能够在一些反式作用因子的辅助下,招募核糖体小亚基到病毒mRNA的翻译起始位点.前,依赖IRES元件翻译起始的RNA病毒在哺乳动物,无脊椎动物及植物中均有发现.因此,对RNA病毒IRES元件的深入研究,不仅有助于阐明相关疾病的发生机理,而且为工业应用和疾病治疗提供借鉴意义.本文对RNA病毒IRES元件发现、分类、结构与功能等作了综述.     

Bridging IRES elements in mRNAs to the eukaryotic translation apparatus

IRES elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, ribosome-scanning model, the mechanism of IRES-mediated translation initiation is not well understood. IRES elements, first discovered in viral RNA genomes, were subsequently found in a subset of cellular RNAs as well. Interestingly, these cellular IRES-containing mRNAs appear to play important roles during conditions of cellular stress, development, and disease (e.g., cancer). It has been shown for viral IRESes that some require specific IRES trans-acting factors (ITAFs), while others require few if any additional proteins and can bind ribosomes directly. Current studies are aimed at elucidating the mechanism of IRES-mediated translation initiation and features that may be common or differ greatly among cellular and viral IRESes. This review will explore IRES elements as important RNA structures that function in both cellular and viral RNA translation and the significance of these structures in providing an alternative mechanism of eukaryotic translation initiation.     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

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.     

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.     

Molecular Biology and Cell-free Synthesis of Poliovirus

Abstract. Poliovirus is a small icosahedral particle consisting of only five species of macromolecules: 60 copies each of the capsid protein VP1-4; and one copy of single-stranded RNA, approximately 7500 nt long. The genome, linked at the 5′ end to a small protein VPg and 3′ polyadenylylated, is of plus strand polarity. After receptor-mediated uptake of the virus and release of the RNA into the cytoplasm, the genome serves as mRNA, encoding only a single polypeptide, the polyprotein. The polyprotein is cleaved co-translationally into numerous polypeptides by its own, internal proteinases 2A^pro, 3C^pro and 3CD^pro. Initiation of translation is mediated by a novel genetic element, called internal ribosomal entry site (IRES). IRES elements, which are 400 nt long RNA segments located within the 5′ non-translated region of the viral genome, are common to all picornaviruses. Their function renders translation of picornavirus mRNAs cap- and 5′-independent, an observation that has upset the dogma of cap-dependent translation in eukaryotic cells. IRES elements have also been used to genetically dissect the viral genome and to construct novel expression vectors. Genome replication is not fully understood, the major conundrum being the initiation of RNA synthesis by the primer-dependent viral RNA polymerase 3D^pol, a process leading to VPg-linked RNA products. Nearly all non-structural proteins appear to be involved in initiation, the proteinases 2A^pro and 3CD^pro included. A HeLa cell-free system has been developed that, on programming with plasmid-transcribed viral RNA, will perform viral translation, protein processing, RNA replication, and assembly of capsid protein and newly made genomic RNA. The final yield is infectious poliovirus. This result has nullified the dictum that no virus can replicate in a cell-free medium.     

A Six-Nucleotide Segment within the 3′ Untranslated Region of Hibiscus Chlorotic Ringspot Virus Plays an Essential Role in Translational Enhancement

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3' untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3' UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3' region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3' UTR.     

Selection and analysis of mutations in an encephalomyocarditis virus internal ribosome entry site that improve the efficiency of a bicistronic flavivirus construct

Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3' noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.