The pathway of HCV IRES-mediated translation initiation

The HCV internal ribosome entry site (IRES) directly regulates the assembly of translation initiation complexes on viral mRNA by a sequential pathway that is distinct from canonical eukaryotic initiation. The HCV IRES can form a binary complex with an eIF-free 40S ribosomal subunit. Next, a 48S-like complex assembles at the AUG initiation codon upon association of eIF3 and ternary complex. 80S complex formation is rate limiting and follows the GTP-dependent association of the 60S subunit. Efficient assembly of the 48S-like and 80S complexes on the IRES mRNA is dependent upon maintenance of the highly conserved HCV IRES structure. This revised model of HCV IRES translation initiation provides a context to understand the function of different HCV IRES domains during translation initiation.     

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.     

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.     

Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES

Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.     

HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.     

Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.     

Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.     

Affinity purification of eukaryotic 48S initiation complexes

In vitro assembly of translation initiation complexes from higher eukaryotes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources, and therefore yields only limited material for functional and structural studies. Here we describe a robust, affinity chromatography-based purification of eukaryotic 48S initiation complexes from rabbit reticulocyte lysate (RRL), which significantly reduces the number of individual purification steps. Hybrid RNA molecules, consisting of either a canonical 5' UTR or an internal ribosome entry site (IRES) RNA followed by a short open reading frame and a streptomycin aptamer sequence, are incubated in RRL to form 48S complexes. The assembly reaction is then applied to a dihydrostreptomycin-sepharose column; bound complexes are washed and specifically eluted upon addition of streptomycin. The eluted fractions are further purified by centrifugation through a sucrose density gradient to yield pure 48S particles. Using this purification scheme, properly assembled IRES-mediated as well as canonical 48S complexes were purified in milligram quantities.     

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.     

Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites

Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated approximately 300 nt.-long Hepatitis C virus (HCV) IRES and the approximately 200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine-Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.     

eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II

Specific interactions of the classical swine fever virus internal ribosomal entry site (IRES) with 40S ribosomal subunits and eukaryotic translation initiation factor (eIF)3 enable 43S preinitiation complexes containing eIF3 and eIF2-GTP-Met-tRNA(iMet) to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding to IRES-40S complexes, forming 48S complexes that can assemble elongation-competent ribosomes. Although 48S complexes assembled both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(iMet) recruitment were destabilized by eIF1, dissociation of 48S complexes formed with eIF2 could be out-competed by efficient subunit joining. Deletion of IRES domain II, which is responsible for conformational changes induced in 40S subunits by IRES binding, eliminated the sensitivity of 48S complexes assembled by eIF2/eIF3- and eIF5B/eIF3-mediated mechanisms to eIF1-induced destabilization. However, 48S complexes formed by the eIF5B/eIF3-mediated mechanism on the truncated IRES could not undergo efficient subunit joining, as reported previously for analogous complexes assembled with eIF2, indicating that domain II is essential for general conformational changes in 48S complexes, irrespective of how they were assembled, that are required for eIF5-induced hydrolysis of eIF2-bound GTP and/or subunit joining.     

Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1

Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).     

Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.

The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested.     

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.     

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.     

An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.     

Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions.

Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (p52, p57, p70, p72, and p100) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, p57, binding to the IRES correlates with translation. Recently, p57 was identified to be very similar, if not identical, to polypyrimidine tract-binding protein. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for p52, p57, p70, and p100 are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.     

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.