Role of RNA Structure Motifs in IRES-Dependent Translation Initiation of the Coxsackievirus B3: New Insights for Developing Live-Attenuated Strains for Vaccines and Gene Therapy

Internal ribosome entry site (IRES) elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, the mechanism of IRES-mediated translation initiation is still poorly understood. Translation initiation of the coxsackievirus B3 (CVB3), a causative agent of viral myocarditis, has been shown to be mediated by a highly ordered structure of the 5′ untranslated region (5′UTR), which harbors an IRES. Taking into account that efficient initiation of mRNA translation depends on temporally and spatially orchestrated sequence of RNA–protein and RNA–RNA interactions, and that, at present, little is known about these interactions, we aimed to describe recent advances in our understanding of molecular structures and biochemical functions of the translation initiation process. Thus, this review will explore the IRES elements as important RNA structures and the significance of these structures in providing an alternative mechanism of translation initiation of the CVB3 RNA. Since translation initiation is the first intracellular step during the CVB3 infection cycle, the IRES region provides an ideal target for antiviral therapies. Interestingly, the 5′ and 3′UTRs represent promising candidates for the study of CVB3 cardiovirulence and provide new insights for developing live-attenuated vaccines.     

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.     

Inhibition of hepatitis C virus IRES-mediated translation by small RNAs analogous to stem-loop structures of the 5'-untranslated region

Translation of the hepatitis C virus (HCV) RNA is mediated by the interaction of ribosomes and cellular proteins with an internal ribosome entry site (IRES) located within the 5′-untranslated region (5′-UTR). We have investigated whether small RNA molecules corresponding to the different stem–loop (SL) domains of the HCV IRES, when introduced in trans, can bind to the cellular proteins and antagonize their binding to the viral IRES, thereby inhibiting HCV IRES-mediated translation. We have found that a RNA molecule corresponding to SL III could efficiently inhibit HCV IRES-mediated translation in a dose-dependent manner without affecting cap-dependent translation. The SL III RNA was found to bind to most of the cellular proteins which interacted with the HCV 5′-UTR. A smaller RNA corresponding to SL e+f of domain III also strongly and selectively inhibited HCV IRES-mediated translation. This RNA molecule interacted with the ribosomal S5 protein and prevented the recruitment of the 40S ribosomal subunit. This study reveals valuable insights into the role of the SL structures of the HCV IRES in mediating ribosome entry. Finally, these results provide a basis for developing anti-HCV therapy using small RNA molecules mimicking the SL structures of the 5′-UTR to specifically block viral RNA translation.     

IRESdb: the Internal Ribosome Entry Site database

Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon. These sequences are very diverse and are present in a growing list of mRNAs. Novel IRES sequences continue to be added to public databases every year and the list of unknown IRESes is certainly still very large. The IRES database is a comprehensive WWW resource for internal ribosome entry sites and presents currently available general information as well as detailed data for each IRES. It is a searchable, periodically updated collection of IRES RNA sequences. Sequences are presented in FASTA form and hotlinked to NCBI GenBank files. Several subsets of data are classified according to the viral taxon (for viral IRESes), to the gene product function (for cellular IRESes), to the possible cellular regulation or to the trans-acting factor that mediates IRES function. This database is accessible at http://ifr31w3.toulouse.inserm.fr/IRESdatabase/.     

Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem–loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA^Phe or its anticodon stem– loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.     

Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.     

Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements

We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.     

RNA structure-based ribosome recruitment: lessons from the Dicistroviridae intergenic region IRESes

In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5' end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by many viruses of medical and economic importance. In this review, we briefly describe and compare mechanistic and structural groups of viral IRES RNAs, focusing on those IRESes that are capable of direct ribosome recruitment using specific RNA structures. We then discuss in greater detail some recent advances in our understanding of the intergenic region IRESes of the Dicistroviridae, which use the most streamlined ribosome-recruitment mechanism yet discovered. By combining these findings with knowledge of canonical translation and the behavior of other IRESes, mechanistic models of this RNA structure-based process are emerging.     

A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation

A significant number of viral and cellular mRNAs utilize cap-independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap-independent translation requires noncanonical, cellular RNA-binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo-cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)-mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA-binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES-mediated translation), further documenting the multifunctional nature of this important class of cellular RNA-binding proteins.     

Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia

Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.     

Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation

Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.     

A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES

Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.     

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.     

Internal ribosome entry sites of viral and cellular RNAs

In recent years mechanism of internal initation of translation in eukaryotic cells commands the attention of molecular biologists in increasing frequency. Ten years ago, experiments with picornaviruses demonstrated the ability of 40S ribosomal subunits to bind to nucleotide sequences localized far from the 5′ ends of RNA molecules, and since then numerous viral and even cellular RNAs were shown to be capable of internal initiation of translation. In the present survey, data on the localization, structure, and functional load of these internal ribosome entry sites (IRES elements) of viral and cellular RNAs, as well as on proteins capable of strong and highly specific binding to IRES elements, are discussed. A conclusion is that a unified model of structure and fuctioning of viral and cellular IRES elements cannot be suggested.     

Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.     

The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.     

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.